Fusion proteins and methods for treating, preventing or ameliorating pain

ABSTRACT

A single chain, polypeptide fusion protein, comprising: a non-cytotoxic protease, which protease is capable of cleaving a protein of the exocytic fusion apparatus of a nociceptive sensory afferent (eg clostridial neurotoxin L-chain or IgA protease); a galanin Targeting Moiety that is capable of binding to a Binding Site on the nociceptive sensory afferent, which Binding Site is capable of undergoing endocytosis to be incorporated into an endosome within the nociceptive sensory afferent (eg GALR1, GALR2, or GALR3 receptor); a protease cleavage site at which site the fusion protein is cleavable by a protease, wherein the protease cleavage site is located between the non-cytotoxic protease and the galanin Targeting Moiety; a translocation domain that is capable of translocating the protease from within an endosome, across the endosomal membrane and into the cytosol of the nociceptive sensory afferent (eg HN domain of clostridial neurotoxin); a first spacer located between the non-cytotoxic protease and the protease cleavage site, wherein said first spacer comprises an amino acid sequence of from 4 to 25 amino acid residues; and a second spacer located between the galanin Targeting Moiety and the translocation domain, wherein said second spacer comprises an amino acid sequence of from 4 to 35 amino acid residues. Nucleic acid sequences encoding the polypeptide fusion proteins, methods of preparing same and uses thereof are also described (eg of treating, preventing or ameliorating pain).

This invention relates to non-cytotoxic fusion proteins, and to thetherapeutic application thereof as analgesic molecules.

Toxins may be generally divided into two groups according to the type ofeffect that they have on a target cell. In more detail, the first groupof toxins kill their natural target cells, and are therefore known ascytotoxic toxin molecules. This group of toxins is exemplified interalia by plant toxins such as ricin, and abrin, and by bacterial toxinssuch as diphtheria toxin, and Pseudomonas exotoxin A. Cytotoxic toxinshave attracted much interest in the design of “magic bullets” (e.g.immunoconjugates, which comprise a cytotoxic toxin component and anantibody that binds to a specific marker on a target cell) for thetreatment of cellular disorders and conditions such as cancer. Cytotoxictoxins typically kill their target cells by inhibiting the cellularprocess of protein synthesis.

The second group of toxins, which are known as non-cytotoxic toxins, donot (as their name confirms) kill their natural target cells.Non-cytotoxic toxins have attracted much less commercial interest thanhave their cytotoxic counterparts, and exert their effects on a targetcell by inhibiting cellular processes other than protein synthesis.Non-cytotoxic toxins are produced by a variety of plants, and by avariety of microorganisms such as Clostridium sp. and Neisseria sp.

Clostridial neurotoxins are proteins that typically have a molecularmass of the order of 150 kDa. They are produced by various species ofbacteria, especially of the genus Clostridium, most importantly C.tetani and several strains of C. botulinum, C. butyricum and C.argentinense. There are at present eight different classes of theclostridial neurotoxin, namely: tetanus toxin, and botulinum neurotoxinin its serotypes A, B, C1, D, E, F and G, and they all share similarstructures and modes of action.

Clostridial neurotoxins represent a major group of non-cytotoxic toxinmolecules, and are synthesised by the host bacterium as singlepolypeptides that are modified post-translationally by a proteolyticcleavage event to form two polypeptide chains joined together by adisulphide bond. The two chains are termed the heavy chain (H-chain),which has a molecular mass of approximately 100 kDa, and the light chain(L-chain or LC), which has a molecular mass of approximately 50 kDa.

L-chains possess a protease function (zinc-dependent endopeptidaseactivity) and exhibit a high substrate specificity for vesicle and/orplasma membrane associated proteins involved in the exocytic process.L-chains from different clostridial species or serotypes may hydrolysedifferent but specific peptide bonds in one of three substrate proteins,namely synaptobrevin, syntaxin or SNAP-25. These substrates areimportant components of the neurosecretory machinery.

Neisseria sp., most importantly from the species N. gonorrhoeae, producefunctionally similar non-cytotoxic proteases. An example of such aprotease is IgA protease (see WO99/58571).

It has been well documented in the art that toxin molecules may bere-targeted to a cell that is not the toxin's natural target cell. Whenso re-targeted, the modified toxin is capable of binding to a desiredtarget cell and, following subsequent translocation into the cytosol, iscapable of exerting its effect on the target cell. Said re-targeting isachieved by replacing the natural Targeting Moiety (TM) of the toxinwith a different TM. In this regard, the TM is selected so that it willbind to a desired target cell, and allow subsequent passage of themodified toxin into an endosome within the target cell. The modifiedtoxin also comprises a translocation domain to enable entry of thenon-cytotoxic protease into the cell cytosol. The translocation domaincan be the natural translocation domain of the toxin or it can be adifferent translocation domain obtained from a microbial protein withtranslocation activity.

The above-mentioned TM replacement may be effected by conventionalchemical conjugation techniques, which are well known to a skilledperson. In this regard, reference is made to Hermanson, G. T. (1996),Bioconjugate techniques, Academic Press, and to Wong, S. S. (1991),Chemistry of protein conjugation and cross-linking, CRC Press.Alternatively, recombinant techniques may be employed, such as thosedescribed in WO98/07864. All of the above cited references areincorporated by reference herein.

Pain-sensing cells possess a wide range of receptor types. However, notall receptor types are suited (least of all desirable) forreceptor-mediated endocytosis. Similarly, binding properties can varywidely between different TMs for the same receptor, and even more sobetween different TMs and different receptors.

There is therefore a need to develop modified non-cytotoxic fusionproteins that address one or more of the above problems. Of particularinterest is the development of an alternative/improved non-cytotoxicfusion protein for use in treating pain.

The present invention seeks to address one or more of the above problemsby providing unique fusion proteins.

The present invention addresses one or more of the above-mentionedproblems by providing a single chain, polypeptide fusion protein,comprising:

-   -   a. a non-cytotoxic protease which protease cleaves a protein of        the exocytic fusion apparatus of a nociceptive sensory afferent;    -   b. a galanin Targeting Moiety that binds to a Binding Site on        the nociceptive sensory afferent, which Binding Site endocytoses        to be incorporated into an endosome within the nociceptive        sensory afferent;    -   c. a protease cleavage site at which site the fusion protein is        cleavable by a protease, wherein the protease cleavage site is        located between the non-cytotoxic protease and the galanin        Targeting Moiety;    -   d. a translocation domain that translocates the protease from        within an endosome, across the endosomal membrane and into the        cytosol of the nociceptive sensory afferent, wherein the        Targeting Moiety is located between the protease cleavage site        and the translocation domain;    -   e. a first spacer located between the non-cytotoxic and the        protease cleavage site, wherein said first spacer comprises an        amino acid sequence of from 4 to 25 amino acid residues;    -   f. a second spacer located between the galanin Targeting Moiety        and the translocation domain, wherein said second spacer        comprises an amino acid sequence of from 4 to 35 amino acid        residues.

The non-cytotoxic protease component of the present invention is anon-cytotoxic protease, which protease is capable of cleaving differentbut specific peptide bonds in one of three substrate proteins, namelysynaptobrevin, syntaxin or SNAP-25, of the exocytic fusion apparatus ina nociceptive sensory afferent. These substrates are importantcomponents of the neurosecretory machinery. The non-cytotoxic proteasecomponent of the present invention is preferably a neisserial IgAprotease or a clostridial neurotoxin L-chain. The term non-cytotoxicprotease embraces functionally equivalent fragments and derivatives ofsaid non-cytotoxic protease(s). A particularly preferred non-cytotoxicprotease component is a botulinum neurotoxin (BoNT) L-chain.

The translocation component of the present invention enablestranslocation of the non-cytotoxic protease (or fragment thereof) intothe target cell such that functional expression of protease activityoccurs within the cytosol of the target cell. The translocationcomponent is preferably capable of forming ion-permeable pores in lipidmembranes under conditions of low pH. Preferably it has been found touse only those portions of the protein molecule capable ofpore-formation within the endosomal membrane. The translocationcomponent may be obtained from a microbial protein source, in particularfrom a bacterial or viral protein source. Hence, in one embodiment, thetranslocation component is a translocating domain of an enzyme, such asa bacterial toxin or viral protein. The translocation component of thepresent invention is preferably a clostridial neurotoxin H-chain or afragment thereof. Most preferably it is the H_(N) domain (or afunctional component thereof), wherein H_(N) means a portion or fragmentof the H-chain of a clostridial neurotoxin approximately equivalent tothe amino-terminal half of the H-chain, or the domain corresponding tothat fragment in the intact H-chain.

The galanin TM component of the present invention is responsible forbinding the fusion protein of the present invention to a Binding Site ona target cell. Thus, the galanin TM component is a ligand through whichthe fusion proteins of the present invention bind to a selected targetcell.

In the context of the present invention, the target cell is anociceptive sensory afferent, preferably a primary nociceptive afferent(e.g. an A-fibre such as an Aδ-fibre or a C-fibre). Thus, the fusionproteins of the present invention are capable of inhibitingneurotransmitter or neuromodulator [e.g. glutamate, substance P,calcitonin-gene related peptide (CGRP), and/or neuropeptide Y] releasefrom discrete populations of nociceptive sensory afferent neurons. Inuse, the fusion proteins reduce or prevent the transmission of sensoryafferent signals (e.g. neurotransmitters or neuromodulators) fromperipheral to central pain fibres, and therefore have application astherapeutic molecules for the treatment of pain, in particular chronicpain.

It is routine to confirm that a TM binds to a nociceptive sensoryafferent. For example, a simple radioactive displacement experiment maybe employed in which tissue or cells representative of the nociceptivesensory afferent (for example DRGs) are exposed to labelled (e.g.tritiated) ligand in the presence of an excess of unlabelled ligand. Insuch an experiment, the relative proportions of non-specific andspecific binding may be assessed, thereby allowing confirmation that theligand binds to the nociceptive sensory afferent target cell.Optionally, the assay may include one or more binding antagonists, andthe assay may further comprise observing a loss of ligand binding.Examples of this type of experiment can be found in Hulme, E. C. (1990),Receptor-binding studies, a brief outline, pp. 303-311, In Receptorbiochemistry, A Practical Approach, Ed. E. C. Hulme, Oxford UniversityPress.

The fusion proteins of the present invention generally demonstrate areduced binding affinity (in the region of up to 10-fold) for thegalanin receptor (e.g. GALR1) when compared with the corresponding‘free’ TM (e.g. gal16). However, despite this observation, the fusionproteins of the present invention surprisingly demonstrate goodefficacy. This can be attributed to two principal features. First, thenon-cytotoxic protease component is catalytic—thus, the therapeuticeffect of a few such molecules is rapidly amplified. Secondly, thegalanin receptors present on the nociceptive sensory afferents need onlyact as a gateway for entry of the therapeutic, and need not necessarilybe stimulated to a level required in order to achieve a ligand-receptormediated pharmacological response. Accordingly, the fusion proteins ofthe present invention may be administered at a dosage that is much lowerthat would be employed for other types of analgesic molecules such asNSAIDS, morphine, and gabapentin. The latter molecules are typicallyadministered at high microgram to milligram (even up to hundreds ofmilligram) quantities, whereas the fusion proteins of the presentinvention may be administered at much lower dosages, typically at least10-fold lower, and more typically at 100-fold lower.

The galanin TM of the invention can also be a molecule that acts as an“agonist” at one or more of the galanin receptors present on anociceptive sensory afferent, more particularly on a primary nociceptiveafferent. Conventionally, an agonist has been considered any moleculethat can either increase or decrease activities within a cell, namelyany molecule that simply causes an alteration of cell activity. Forexample, the conventional meaning of an agonist would include a chemicalsubstance capable of combining with a receptor on a cell and initiatinga reaction or activity, or a drug that induces an active response byactivating receptors, whether the response is an increase or decrease incellular activity.

However, for the purposes of this invention, an agonist is morespecifically defined as a molecule that is capable of stimulating theprocess of exocytic fusion in a target cell, which process issusceptible to inhibition by a protease (or fragment thereof) capable ofcleaving a protein of the exocytic fusion apparatus in said target cell.

Accordingly, the particular agonist definition of the present inventionwould exclude many molecules that would be conventionally considered asagonists. For example, nerve growth factor (NGF) is an agonist inrespect of its ability to promote neuronal differentiation via bindingto a TrkA receptor. However, NGF is not an agonist when assessed by theabove criteria because it is not a principal inducer of exocytic fusion.In addition, the process that NGF stimulates (i.e. cell differentiation)is not susceptible to inhibition by the protease activity of anon-cytotoxic toxin molecule.

In one embodiment, the fusion proteins according to the presentinvention demonstrate preferential receptor binding and/orinternalisation properties. This, in turn, may result in more efficientdelivery of the protease component to a pain-sensing target cell.

Use of an agonist as a TM is self-limiting with respect to side-effects.In more detail, binding of an agonist TM to a pain-sensing target cellincreases exocytic fusion, which may exacerbate the sensation of pain.However, the exocytic process that is stimulated by agonist binding issubsequently reduced or inhibited by the protease component of thefusion protein.

The agonist properties of a TM that binds to a receptor on a nociceptiveafferent can be confirmed using the methods described in Example 9.

The Targeting Moiety of the present invention comprises or consists ofgalanin and/or derivatives of galanin. Galanin receptors (e.g. GALR1,GALR2 and GALR3) are found pre- and post-synaptically in DRGs (Liu &Hokfelt, (2002), Trends Pharm. Sci., 23(10), 468-74), and are enhancedin expression during neuropathic pain states. Xu et al., (2000)Neuropeptides, 34 (3&4), 137-147 provides further information inrelation to galanin. All of the above cited references are incorporatedby reference herein.

In one embodiment of the invention, the target for the galanin TM is theGALR1, GALR2 and/or the GALR3 receptor. These receptors are members ofthe G-protein-coupled class of receptors, and have a seven transmembranedomain structure.

In one embodiment, the galanin TM is a molecule that binds (preferablythat specifically binds) to the GALR1, GALR2 and/or the GALR3 receptor.More preferably, the galanin TM is an “agonist” of the GALR1, GALR2and/or the GALR3 receptor. The term “agonist” in this context is definedas above.

Wild-type human galanin peptide is a 30 amino acid peptide, abbreviatedherein as “GA30” (represented by SEQ ID NO: 7). In one embodiment, thegalanin TM comprises or consists of SEQ ID NO: 7.

The invention also encompasses fragments, variants, and derivatives ofthe galanin TM described above. These fragments, variants, andderivatives substantially retain the properties that are ascribed tosaid galanin TM (i.e. are functionally equivalent). For example, thefragments, variants, and derivatives may retain the ability to bind tothe GALR1, GALR2 and/or GALR3 receptor. In one embodiment, the galaninTM of the invention comprises or consists of a 16 amino acid fragment offull-length galanin peptide and is referred to herein as GA16(represented by SEQ ID NO: 8).

In one embodiment, the galanin TM comprises or consists of an amino acidsequence having at least 70%, preferably at least 80% (such as at least82, 84, 85, 86, 88 or 89%), more preferably at least 90% (such as atleast 91, 92, 93 or 94%), and most preferably at least 95% (such as atleast 96, 97, 98, 99 or 100%) amino acid sequence acid identity to SEQID NO: 7 or SEQ ID NO: 8.

In one embodiment the galanin TM comprises or consists of an amino acidsequence having at least 70% (such as at least 80, 82, 84, 85, 86, 88 or89%), more preferably at least 90% (such as at least 91, 92, 93 or 94%),and most preferably at least 95% (such as at least 96, 97, 98, 99 or100%) amino acid sequence acid identity to full-length amino acidsequence of SEQ ID NO: 7 or SEQ ID NO: 8, or a fragment of SEQ ID NO: 7or SEQ ID NO: 8 comprising or consisting of at least 10 (such as atleast 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28 or 29) contiguous amino acid residues thereof.

In one embodiment, the galanin Targeting Moiety comprises or consists ofan amino acid sequence according to SEQ ID NO. 7 or a fragmentcomprising or consisting of at least 16 (such as at least 10, 11, 12,13, 14 or 15) contiguous amino acid residues thereof, or a variant aminoacid sequence of said SEQ ID NO: 7 or said fragment having a maximum of6 (such as a maximum of 5, 4, 3, 2 or 1) conservative amino acidsubstitutions.

The protease cleavage site of the present invention allows cleavage(preferably controlled cleavage) of the fusion protein at a positionbetween the non-cytotoxic protease component and the TM component. It isthis cleavage reaction that converts the fusion protein from a singlechain polypeptide into a disulphide-linked, di-chain polypeptide.

According to a preferred embodiment of the present invention, thegalanin TM binds via a domain or amino acid sequence that is locatedaway from the C-terminus of the galanin TM. For example, the relevantbinding domain may include an intra domain or an amino acid sequencelocated towards the middle (i.e. of the linear peptide sequence) of theTM. Preferably, the relevant binding domain is located towards theN-terminus of the galanin TM, more preferably at or near to theN-terminus.

In one embodiment, the single chain polypeptide fusion may include morethan one proteolytic cleavage site. However, where two or more suchsites exist, they are different, thereby substantially preventing theoccurrence of multiple cleavage events in the presence of a singleprotease. In another embodiment, it is preferred that the single chainpolypeptide fusion has a single protease cleavage site.

The protease cleavage sequence(s) may be introduced (and/or any inherentcleavage sequence removed) at the DNA level by conventional means, suchas by site-directed mutagenesis. Screening to confirm the presence ofcleavage sequences may be performed manually or with the assistance ofcomputer software (e.g. the MapDraw program by DNASTAR, Inc.).

Whilst any protease cleavage site may be employed, the following arepreferred:

Enterokinase (DDDDK↓) Factor Xa (IEGR↓/IDGR↓) TEV (Tobacco Etch virus)(ENLYFQ↓G) Thrombin (LVPR↓GS) PreScission (LEVLFQ↓GP).

In one embodiment, the protease cleavage site is an enterokinasecleavage site (DDDDK↓). In one embodiment, enterokinase protease is usedto cleave the enterokinase cleavage site and activate the fusionprotein.

Also embraced by the term protease cleavage site is an intein, which isa self-cleaving sequence. The self-splicing reaction is controllable,for example by varying the concentration of reducing agent present.

In use, the protease cleavage site is cleaved and the N-terminal region(preferably the N-terminus) of the TM becomes exposed. The resultingpolypeptide has a TM with an N-terminal domain or an intra domain thatis substantially free from the remainder of the fusion protein. Thisarrangement ensures that the N-terminal component (or intra domain) ofthe TM may interact directly with a Binding Site on a target cell.

In one embodiment, the TM and the protease cleavage site are distancedapart in the fusion protein by at most 10 amino acid residues, morepreferably by at most 5 amino acid residues, and most preferably by zeroamino acid residues. In one embodiment, the TM and the protease cleavagesite are distanced apart in the fusion protein by 0-10 (such as 0-9,0-8, 0-7, 0-6, 0-5, 0-4, 0-3, 0-2) and preferably 0-1 amino acidresidues Thus, following cleavage of the protease cleavage site, afusion is provided with a TM that has an N-terminal domain that issubstantially free from the remainder of the fusion. This arrangementensures that the N-terminal component of the Targeting Moiety mayinteract directly with a Binding Site on a target cell.

One advantage associated with the above-mentioned activation step isthat the TM only becomes susceptible to N-terminal degradation onceproteolytic cleavage of the fusion protein has occurred. In addition,the selection of a specific protease cleavage site permits selectiveactivation of the polypeptide fusion into a di-chain conformation.

Construction of the single-chain polypeptide fusion of the presentinvention places the protease cleavage site between the TM and thenon-cytotoxic protease component.

It is preferred that, in the single-chain fusion, the TM is locatedbetween the protease cleavage site and the translocation component. Thisensures that the TM is attached to the translocation domain (i.e. asoccurs with native clostridial holotoxin), though in the case of thepresent invention the order of the two components is reversed vis-à-visnative holotoxin. A further advantage with this arrangement is that theTM is located in an exposed loop region of the fusion protein, which hasminimal structural effects on the conformation of the fusion protein. Inthis regard, said loop is variously referred to as the linker, theactivation loop, the inter-domain linker, or just the surface exposedloop (Schiavo et al 2000, Phys. Rev., 80, 717-766; Turton et al., 2002,Trends Biochem. Sci., 27, 552-558).

The single chain fusion protein of the present invention comprises afirst spacer located between the non-cytotoxic protease and the proteasecleavage site, wherein said first spacer comprises (or consists of) anamino acid sequence of from 4 to 25 (such as from 6 to 25, 8 to 25, 10to 25, 15 to 25 or from 4 to 21, 4 to 20, 4 to 18, 4 to 15, 4 to 12 or 4to 10) amino acid residues. In one embodiment, the first spacercomprises (or consists of) an amino acid sequence of at least 4 (such asat least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) amino acid residues. Inone embodiment, the first spacer comprises (or consists of) an aminoacid sequence of at most 25 (such as at most 24, 23, 22, 21, 20, 19, 18,17, 16, 15, 14, 13, 12, 10) amino acid residues. Said first spacerenables cleavage of the fusion protein at the protease cleavage site.

Without a first spacer of the present invention, protease cleavage andactivation of the fusion protein is markedly poor. Without wishing to bebound by theory, it is hypothesised that the galanin Targeting Moietymay sterically block or interact with the protease cleavage siteresulting in poor activation of fusion proteins lacking a first spacerof the present invention. The present inventors believe that it is theflexibility afforded by the first spacer which provides for theenhanced/improved activation properties of the presently claimed fusionproteins. Rigid linkers such as alpha-helical linkers do not afford thenecessary flexibility. This is also true for galanin fusion proteinshaving ‘natural’ spacer sequences containing a protease cleavage site,which may replicate undesirable rigid alpha-helical linker structures.Flexibility and mobility of polypeptide domains can be ascertained by anumber of methods including determining the X-ray crystallographicB-factor (see e.g. Smith et al., 2003 Protein Science, 12:1060-1072;incorporated by reference herein). The specifically selected spacersequences of the present invention provide for enhanced activation overand above any ‘natural’ spacer sequences. Activation in this contextmeans that said first spacer enables cleavage of the fusion protein atthe protease cleavage site. Particularly preferred amino acid residuesfor use in the first spacer include glycine, threonine, arginine,serine, alanine, asparagine, glutamine, aspartic acid, proline, glutamicacid and/or lysine. The aforementioned amino acids are considered to bethe most flexible amino acids—see Smith et al. 2003 Protein Science2003; 12:1060-1072.

In one embodiment, the amino acid residues of the first spacer areselected from the group consisting of glycine, threonine, arginine,serine, asparagine, glutamine, alanine, aspartic acid, proline, glutamicacid, lysine, leucine and/or valine. In one embodiment, the amino acidresidues of the first spacer are selected from the group consisting ofglycine, serine, alanine, leucine and/or valine. In one embodiment, theamino acid residues of the first spacer are selected from the groupconsisting of glycine, serine and/or alanine. Glycine and serine areparticularly preferred. In one embodiment, the first spacer comprises orconsists of one or more pentapeptides having glycine, serine, and orthreonine residues. One way of assessing whether the first spacerpossesses the requisite flexibility in the presently claimed fusionproteins is by performing a simple protease cleavage assay. It would beroutine for a person skilled in the art to assess cleavage/activation ofa fusion protein—standard methodology is described, for example, inExample 1.

In one embodiment, the first spacer may be selected from a GS5, GS10,GS15, GS18, GS20, FL3 and/or FL4 spacers. The sequence of said spacersis provided in Table 1, below.

TABLE 1 Spacer Sequence GS5 GGGGSA GS10 GGGGSGGGGSA GS15ALAGGGGSGGGGSALV GS18 GGGGSGGGGSGGGGSA GS20 ALAGGGGSGGGGSGGGGSALV FL3LGGGGSGGGGSGGGGSAAA FL4 LSGGGGSGGGGSGGGGSGGGGSAAA

In one embodiment, the first spacer enables at least 45% (such as atleast 50, 55, 60, 65, 70, 75, 80, 90, 95, 98, 99 or 100%) activation ofthe fusion protein by protease cleavage. In one embodiment, the firstspacer enables at least 70% activation of the fusion protein by proteasecleavage.

In one embodiment, the first spacer is not a naturally-occurring spacersequence. In one embodiment, the first spacer does not comprise orconsist of an amino acid sequence native to the natural (i.e. wild-type)clostridial neurotoxin, such as botulinum neurotoxin. In other words,the first spacer may be a non-clostridial sequence (i.e. not found inthe native clostridial neurotoxin). In one embodiment, the fusionprotein does not comprise or consist of the amino acid sequence GIITSK(BoNT/A); VK (BoNT B); AIDGR (BoNT/C); LTK (BoNT/D); IVSVK (BoNT/E);VIPR (BONT/F); VMYK (BoNT/G) and/or IIPPTNIREN (TeNT) as the firstspacer.

In one embodiment, the first spacer begins on the third amino acidresidue following the conserved cysteine residue in the clostridialneurotoxin L-chain (see Table 3 below). In one embodiment, the firstspacer begins after the VD amino acid residues of a non-cytotoxicprotease clostridial L-chain engineered with a sal1 site following theconserved cysteine residue. In one embodiment, the first spacer endswith the amino acid residue marking the beginning of the proteasecleavage sites mentioned above.

In one embodiment, the single chain fusion protein comprises a secondspacer, which is located between the galanin Targeting Moiety and thetranslocation domain. Said second spacer may comprise (or consist of) anamino acid sequence of from 4 to 35 (such as from 6 to 35, 10 to 35, 15to 35, 20 to 35 or from 4 to 28, 4 to 25, 4 to 20 or 4 to 10) amino acidresidues. The present inventors have unexpectedly found that the fusionproteins of the present invention may demonstrate an improved bindingactivity when the size of the second spacer is selected so that (in use)the C-terminus of the TM and the N-terminus of the translocationcomponent are separated from one another by 40-105 angstroms, preferablyby 50-100 angstroms, and more preferably by 50-90 angstroms.

Suitable second spacers may be routinely identified and obtainedaccording to Crasto, C. J. and Feng, J. A. (2000) May, 13(5), pp.309-312—see also http://www.fccc./edu/research/labs/feng/limker.html. Inone embodiment, the second spacer is selected from a GS5, GS10, GS15,GS18, GS20 or HX27 spacer. The sequence of said spacers is provided inTable 2, below.

TABLE 2 Spacer Sequence GS5 GGGGSA GS10 GGGGSGGGGSA GS15ALAGGGGSGGGGSALV GS18 GGGGSGGGGSGGGGSA GS20 ALAGGGGSGGGGSGGGGSALV HX27ALAAEAAAKEAAAKEAAAKAGGGGSALV

The Inventors have surprisingly found, that the presently claimed fusionproteins having said first and second spacer features display enhancedactivation properties and increased yield during recombinant expression.In addition, the presently claimed fusion proteins display enhancedpotency compared to fusion proteins wherein the galanin TM is C-terminalof the translocation domain component.

In one embodiment, the invention provides a single-chain polypeptidefusion protein comprising (or consisting of) an amino acid sequencehaving at least 80% (such as at least 85, 90, 92, 94, 95, 96, 97, 98, 99or 100%) sequence identity to the amino acid sequence of SEQ ID NOs: 10,11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 53, 56and/or 59.

In one embodiment, the invention provides a single-chain polypeptidefusion protein comprising (or consisting of) an amino acid sequencehaving at least 80% (such as at least 85, 90, 92, 94, 95, 96, 97, 98, 99or 100%) sequence identity to the full-length amino acid sequence of SEQID NOs: 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,53, 56 and/or 59.

In one embodiment, in the single chain polypeptide, the non-cytotoxicprotease component and the translocation component are linked togetherby a disulphide bond. Thus, following cleavage of the protease cleavagesite, the polypeptide assumes a di-chain conformation, wherein theprotease and translocation components remain linked together by thedisulphide bond. To this end, it is preferred that the protease andtranslocation components are distanced apart from one another in thesingle chain fusion protein by a maximum of 100 amino acid residues,more preferably a maximum of 80 amino acid residues, particularlypreferably by a maximum of 60 amino acid residues, and most preferablyby a maximum of 50 amino acid residues.

In one embodiment, the non-cytotoxic protease component forms adisulphide bond with the translocation component of the fusion protein.For example, the amino acid residue of the protease component that formsthe disulphide bond is located within the last 20, preferably within thelast 10 C-terminal amino acid residues of the protease component.Similarly, the amino acid residue within the translocation componentthat forms the second part of the disulphide bond may be located withinthe first 20, preferably within the first 10 N-terminal amino acidresidues of the translocation component.

Alternatively, in the single chain polypeptide, the non-cytotoxicprotease component and the TM may be linked together by a disulphidebond. In this regard, the amino acid residue of the TM that forms thedisulphide bond is preferably located away from the N-terminus of theTM, more preferably towards to C-terminus of the TM.

In one embodiment, the non-cytotoxic protease component forms adisulphide bond with the TM component of the fusion protein. In thisregard, the amino acid residue of the protease component that forms thedisulphide bond is preferably located within the last 20, morepreferably within the last 10 C-terminal amino acid residues of theprotease component. Similarly, the amino acid residue within the TMcomponent that forms the second part of the disulphide bond ispreferably located within the last 20, more preferably within the last10 C-terminal amino acid residues of the TM.

The above disulphide bond arrangements have the advantage that theprotease and translocation components are arranged in a manner similarto that for native clostridial neurotoxin. By way of comparison,referring to the primary amino acid sequence for native clostridialneurotoxin, the respective cysteine amino acid residues are distancedapart by between 8 and 27 amino acid residues—taken from Popoff, M R &Marvaud, J-C, 1999, Structural & genomic features of clostridialneurotoxins, Chapter 9, in The Comprehensive Sourcebook of BacterialProtein Toxins. Ed. Alouf & Freer:

TABLE 3 ‘Native’ length between Serotype¹ Sequence C—C BoNT/A1CVRGIITSKTKS----LDKGYNKALNDLC 23 BoNT/A2 CVRGIIPFKTKS----LDEGYNKALNDLC23 BoNT/B CKSVKAPG-------------------IC  8 BoNT/CCHKAIDGRS----------LYNKTLDC 15 BoNT/D CLRLTK---------------NSRDDSTC 12BoNT/E CKN-IVSVK----------GIRK---SIC 13 BoNT/FCKS-VIPRK----------GTKAPP-RLC 15 BoNT/G CKPVMYKNT----------GKSE----QC 13TeNT CKKIIPPTNIRENLYNRTASLTDLGGELC 27 ¹Information from proteolyticstrains only

The fusion protein may comprise one or more purification tags, which arelocated N-terminal to the protease component and/or C-terminal to thetranslocation component.

Whilst any purification tag may be employed, the following arepreferred:

His-tag (e.g. 6× histidine), preferably as a C-terminal and/orN-terminal tag

MBP-tag (maltose binding protein), preferably as an N-terminal tag

GST-tag (glutathione-S-transferase), preferably as an N-terminal tag

His-MBP-tag, preferably as an N-terminal tag

GST-MBP-tag, preferably as an N-terminal tag

Thioredoxin-tag, preferably as an N-terminal tag

CBD-tag (Chitin Binding Domain), preferably as an N-terminal tag.

According to a further embodiment of the present invention, one or moreadditional peptide spacer molecules may be included in the fusionprotein. For example, a peptide spacer may be employed between apurification tag and the rest of the fusion protein molecule (e.g.between an N-terminal purification tag and a protease component of thepresent invention; and/or between a C-terminal purification tag and atranslocation component of the present invention.

In accordance with a second aspect of the present invention, there isprovided a DNA sequence that encodes the above-mentioned single chainpolypeptide. In a preferred aspect of the present invention, the DNAsequence is prepared as part of a DNA vector, wherein the vectorcomprises a promoter and terminator.

In a preferred embodiment, the vector has a promoter selected from:

Promoter Induction Agent Typical Induction Condition Tac (hybrid) IPTG0.2 mM (0.05-2.0 mM) AraBAD L-arabinose 0.2% (0.002-0.4%) T7-lacoperator IPTG 0.2 mM (0.05-2.0 mM)

The DNA construct of the present invention is preferably designed insilico, and then synthesised by conventional DNA synthesis techniques.

The above-mentioned DNA sequence information is optionally modified forcodon-biasing according to the ultimate host cell (e.g. E. coli)expression system that is to be employed.

The DNA backbone is preferably screened for any inherent nucleic acidsequence, which when transcribed and translated would produce an aminoacid sequence corresponding to the protease cleave site encoded by thesecond peptide-coding sequence. This screening may be performed manuallyor with the assistance of computer software (e.g. the MapDraw program byDNASTAR, Inc.).

According to a further embodiment of the present invention, there isprovided a method of preparing a non-cytotoxic agent, comprising:

-   -   a. contacting a single-chain polypeptide fusion protein of the        invention with a protease capable of cleaving the protease        cleavage site;    -   b. cleaving the protease cleavage site, and thereby forming a        di-chain fusion protein.

This aspect provides a di-chain polypeptide, which generally mimics thestructure of clostridial holotoxin. In more detail, the resultingdi-chain polypeptide typically has a structure wherein:

-   -   a. the first chain comprises the non-cytotoxic protease, which        protease is capable of cleaving a protein of the exocytic fusion        apparatus of a nociceptive sensory afferent;    -   b. the second chain comprises the galanin TM and the        translocation domain that is capable of translocating the        protease from within an endosome, across the endosomal membrane        and into the cytosol of the nociceptive sensory afferent; and    -   the first and second chains are disulphide linked together.

In one aspect of the invention, the single chain or dich-chainpolypeptide of the invention is for use as medicament/therapeuticmolecule.

In use, the single chain or di-chain polypeptide of the invention treat,prevent or ameliorate pain.

In use, a therapeutically effective amount of a single chain or di-chainpolypeptide of the invention is administered to a patient.

According to a further aspect of the present invention, there isprovided use of a single chain or di-chain polypeptide of the invention,for the manufacture of a medicament for treating, preventing orameliorating pain.

According to a related aspect, there is provided a method of treating,preventing or ameliorating pain in a subject, comprising administeringto said patient a therapeutically effective amount of a single chain ordi-chain polypeptide of the invention.

The compounds described here may be used to treat a patient sufferingfrom one or more types of chronic pain including neuropathic pain,inflammatory pain, headache pain, somatic pain, visceral pain, andreferred pain.

To “treat,” as used here, means to deal with medically. It includes, forexample, administering a compound of the invention to prevent pain or tolessen its severity.

The term “pain,” as used here, means any unpleasant sensory experience,usually associated with a physical disorder. The physical disorder mayor may not be apparent to a clinician. Pain is of two types: chronic andacute. An “acute pain” is a pain of short duration having a suddenonset. One type of acute pain, for example, is cutaneous pain felt oninjury to the skin or other superficial tissues, such as caused by a cutor a burn. Cutaneous nociceptors terminate just below the skin, and dueto the high concentration of nerve endings, produce a well-defined,localized pain of short duration. “Chronic pain” is a pain other than anacute pain. Chronic pain includes neuropathic pain, inflammatory pain,headache pain, somatic pain visceral pain and referred pain.

I. Neuropathic Pain

The compounds of the invention may be used to treat pain caused by orotherwise associated with any of the following neuropathic painconditions.

“Neuropathic pain” means abnormal sensory input, resulting indiscomfort, from the peripheral nervous system, central nervous systems,or both.

A. Symptoms of Neuropathic Pain

Symptoms of neuropathic pain can involve persistent, spontaneous pain,as well as allodynia (a painful response to a stimulus that normally isnot painful), hyperalgesia (an accentuated response to a painfulstimulus that usually causes only a mild discomfort, such as a pinprick), or hyperpathia (where a short discomfort becomes a prolongedsevere pain).

B. Causes of Neuropathic Pain

Neuropathic pain may be caused by any of the following.

1. A traumatic insult, such as, for example, a nerve compression injury(e.g., a nerve crush, a nerve stretch, a nerve entrapment or anincomplete nerve transsection); a spinal cord injury (e.g., ahemisection of the spinal cord); a limb amputation; a contusion; aninflammation (e.g., an inflammation of the spinal cord); or a surgicalprocedure.

2. An ischemic event, including, for example, a stroke and heart attack.

3. An infectious agent

4. Exposure to a toxic agent, including, for example, a drug, analcohol, a heavy metal (e.g., lead, arsenic, mercury), an industrialagent (e.g., a solvent, fumes from a glue) or nitrous oxide.

5. A disease, including, for example, an inflammatory disorder, aneoplastic tumor, an acquired immune deficiency syndrome (AIDS), Lymesdisease, a leprosy, a metabolic disease, a peripheral nerve disorder,like neuroma, a mononeuropathy or a polyneuropathy.

C. Types of Neuropathic Pain

1. Neuralgia.

A neuralgia is a pain that radiates along the course of one or morespecific nerves usually without any demonstrable pathological change inthe nerve structure. The causes of neuralgia are varied. Chemicalirritation, inflammation, trauma (including surgery), compression bynearby structures (for instance, tumors), and infections may all lead toneuralgia. In many cases, however, the cause is unknown orunidentifiable. Neuralgia is most common in elderly persons, but it mayoccur at any age. A neuralgia, includes, without limitation, atrigeminal neuralgia, a post-herpetic neuralgia, a postherpeticneuralgia, a glossopharyngeal neuralgia, a sciatica and an atypicalfacial pain.

Neuralgia is pain in the distribution of a nerve or nerves. Examples aretrigeminal neuralgia, atypical facial pain, and postherpetic neuralgia(caused by shingles or herpes). The affected nerves are responsible forsensing touch, temperature and pressure in the facial area from the jawto the forehead. The disorder generally causes short episodes ofexcruciating pain, usually for less than two minutes and on only oneside of the face. The pain can be described in a variety of ways such as“stabbing,” “sharp,” “like lightning,” “burning,” and even “itchy”. Inthe atypical form of TN, the pain can also present as severe or merelyaching and last for extended periods. The pain associated with TN isrecognized as one the most excruciating pains that can be experienced.

Simple stimuli such as eating, talking, washing the face, or any lighttouch or sensation can trigger an attack (even the sensation of a gentlebreeze). The attacks can occur in clusters or as an isolated attack.

Symptoms include sharp, stabbing pain or constant, burning pain locatedanywhere, usually on or near the surface of the body, in the samelocation for each episode; pain along the path of a specific nerve;impaired function of affected body part due to pain, or muscle weaknessdue to concomitant motor nerve damage; increased sensitivity of the skinor numbness of the affected skin area (feeling similar to a localanesthetic such as a Novacaine shot); and any touch or pressure isinterpreted as pain. Movement may also be painful.

Trigeminal neuralgia is the most common form of neuralgia. It affectsthe main sensory nerve of the face, the trigeminal nerve (“trigeminal”literally means “three origins”, referring to the division of the nerveinto 3 branches). This condition involves sudden and short attacks ofsevere pain on the side of the face, along the area supplied by thetrigeminal nerve on that side. The pain attacks may be severe enough tocause a facial grimace, which is classically referred to as a painfultic (tic douloureux). Sometimes, the cause of trigeminal neuralgia is ablood vessel or small tumor pressing on the nerve. Disorders such asmultiple sclerosis (an inflammatory disease affecting the brain andspinal cord), certain forms of arthritis, and diabetes (high bloodsugar) may also cause trigeminal neuralgia, but a cause is not alwaysidentified. In this condition, certain movements such as chewing,talking, swallowing, or touching an area of the face may trigger a spasmof excruciating pain.

A related but rather uncommon neuralgia affects the glosso-pharyngealnerve, which provides sensation to the throat. Symptoms of thisneuralgia are short, shock-like episodes of pain located in the throat.

Neuralgia may occur after infections such as shingles, which is causedby the varicella-zoster virus, a type of herpesvirus. This neuralgiaproduces a constant burning pain after the shingles rash has healed. Thepain is worsened by movement of or contact with the affected area. Notall of those diagnosed with shingles go on to experience postherpeticneuralgia, which can be more painful than shingles. The pain andsensitivity can last for months or even years. The pain is usually inthe form of an intolerable sensitivity to any touch but especially lighttouch. Postherpetic neuralgia is not restricted to the face; it canoccur anywhere on the body but usually occurs at the location of theshingles rash. Depression is not uncommon due to the pain and socialisolation during the illness.

Postherpetic neuralgia may be debilitating long after signs of theoriginal herpes infection have disappeared. Other infectious diseasesthat may cause neuralgia are syphilis and Lyme disease.

Diabetes is another common cause of neuralgia. This very common medicalproblem affects almost 1 out of every 20 Americans during adulthood.Diabetes damages the tiny arteries that supply circulation to thenerves, resulting in nerve fiber malfunction and sometimes nerve loss.Diabetes can produce almost any neuralgia, including trigeminalneuralgia, carpal tunnel syndrome (pain and numbness of the hand andwrist), and meralgia paresthetica (numbness and pain in the thigh due todamage to the lateral femoral cutaneous nerve). Strict control of bloodsugar may prevent diabetic nerve damage and may accelerate recovery inpatients who do develop neuralgia.

Other medical conditions that may be associated with neuralgias arechronic renal insufficiency and porphyria—a hereditary disease in whichthe body cannot rid itself of certain substances produced after thenormal breakdown of blood in the body. Certain drugs may also cause thisproblem.

2. Deafferentation.

Deafferentation indicates a loss of the sensory input from a portion ofthe body, and can be caused by interruption of either peripheral sensoryfibres or nerves from the central nervous system. A deafferentation painsyndrome, includes, without limitation, an injury to the brain or spinalcord, a post-stroke pain, a phantom pain, a paraplegia, a brachialplexus avulsion injuries, lumbar radiculopathies.

3. Complex Regional Pain Syndromes (CRPSs)

CRPS is a chronic pain syndrome resulting fromsympathetically-maintained pain, and presents in two forms. CRPS 1currently replaces the term “reflex sympathetic dystrophy syndrome”. Itis a chronic nerve disorder that occurs most often in the arms or legsafter a minor or major injury. CRPS 1 is associated with severe pain;changes in the nails, bone, and skin; and an increased sensitivity totouch in the affected limb. CRPS 2 replaces the term causalgia, andresults from an identified injury to the nerve. A CRPS, includes,without limitation, a CRPS Type I (reflex sympathetic dystrophy) and aCRPS Type II (causalgia).

4. Neuropathy.

A neuropathy is a functional or pathological change in a nerve and ischaracterized clinically by sensory or motor neuron abnormalities.

Central neuropathy is a functional or pathological change in the centralnervous system.

Peripheral neuropathy is a functional or pathological change in one ormore peripheral nerves. The peripheral nerves relay information fromyour central nervous system (brain and spinal cord) to muscles and otherorgans and from your skin, joints, and other organs back to your brain.Peripheral neuropathy occurs when these nerves fail to carry informationto and from the brain and spinal cord, resulting in pain, loss ofsensation, or inability to control muscles. In some cases, the failureof nerves that control blood vessels, intestines, and other organsresults in abnormal blood pressure, digestion problems, and loss ofother basic body processes. Risk factors for neuropathy includediabetes, heavy alcohol use, and exposure to certain chemicals anddrugs. Some people have a hereditary predisposition for neuropathy.Prolonged pressure on a nerve is another risk for developing a nerveinjury. Pressure injury may be caused by prolonged immobility (such as along surgical procedure or lengthy illness) or compression of a nerve bycasts, splints, braces, crutches, or other devices. Polyneuropathyimplies a widespread process that usually affects both sides of the bodyequally. The symptoms depend on which type of nerve is affected. Thethree main types of nerves are sensory, motor, and autonomic. Neuropathycan affect any one or a combination of all three types of nerves.Symptoms also depend on whether the condition affects the whole body orjust one nerve (as from an injury). The cause of chronic inflammatorypolyneuropathy is an abnormal immune response. The specific antigens,immune processes, and triggering factors are variable and in many casesare unknown. It may occur in association with other conditions such asHIV, inflammatory bowel disease, lupus erythematosis, chronic activehepatitis, and blood cell abnormalities.

Peripheral neuropathy may involve a function or pathological change to asingle nerve or nerve group (monneuropathy) or a function orpathological change affecting multiple nerves (polyneuropathy).

Peripheral Neuropathies

-   Hereditary disorders    -   Charcot-Marie-Tooth disease    -   Friedreich's ataxia-   Systemic or metabolic disorders    -   Diabetes (diabetic neuropathy)    -   Dietary deficiencies (especially vitamin B-12)    -   Excessive alcohol use (alcoholic neuropathy)    -   Uremia (from kidney failure)    -   Cancer-   Infectious or inflammatory conditions    -   AIDS    -   Hepatitis    -   Colorado tick fever    -   diphtheria    -   Guillain-Barre syndrome    -   HIV infection without development of AIDS    -   leprosy    -   Lyme    -   polyarteritis nodosa    -   rheumatoid arthritis    -   sarcoidosis    -   Sjogren syndrome    -   syphilis    -   systemic lupus erythematosus    -   amyloid-   Exposure to toxic compounds    -   sniffing glue or other toxic compounds    -   nitrous oxide    -   industrial agents—especially solvents    -   heavy metals (lead, arsenic, mercury, etc.)    -   Neuropathy secondary to drugs like analgesic nephropathy-   Miscellaneous causes    -   ischemia (decreased oxygen/decreased blood flow)    -   prolonged exposure to cold temperature    -   a. Polyneuropathy    -   Polyneuropathy is a peripheral neuropathy involving the loss of        movement or sensation to an area caused by damage or destruction        to multiple peripheral nerves. Polyneuropathic pain, includes,        without limitation, post-polio syndrome, postmastectomy        syndrome, diabetic neuropathy, alcohol neuropathy, amyloid,        toxins, AIDS, hypothyroidism, uremia, vitamin deficiencies,        chemotherapy-induced pain, 2′,3′-didexoycytidine (ddC)        treatment, Guillain-Barré syndrome or Fabry's disease.    -   b. Mononeuropathy    -   Mononeuropathy is a peripheral neuropathy involving loss of        movement or sensation to an area caused by damage or destruction        to a single peripheral nerve or nerve group. Mononeuropathy is        most often caused by damage to a local area resulting from        injury or trauma, although occasionally systemic disorders may        cause isolated nerve damage (as with mononeuritis multiplex).        The usual causes are direct trauma, prolonged pressure on the        nerve, and compression of the nerve by swelling or injury to        nearby body structures. The damage includes destruction of the        myelin sheath (covering) of the nerve or of part of the nerve        cell (the axon). This damage slows or prevents conduction of        impulses through the nerve. Mononeuropathy may involve any part        of the body. Mononeuropathic pain, includes, without limitation,        a sciatic nerve dysfunction, a common peroneal nerve        dysfunction. a radial nerve dysfunction, an ulnar nerve        dysfunction, a cranial mononeuropathy VI, a cranial        mononeuropathy VII, a cranial mononeuropathy III (compression        type), a cranial mononeuropathy III (diabetic type), an axillary        nerve dysfunction, a carpal tunnel syndrome, a femoral nerve        dysfunction, a tibial nerve dysfunction, a Bell's palsy, a        thoracic outlet syndrome, a carpal tunnel syndrome and a sixth        (abducent) nerve palsy    -   c. Generalized peripheral neuropathies    -   Generalized peripheral neuropathis are symmetrical, and usually        due to various systematic illnesses and disease processes that        affect the peripheral nervous system in its entirety. They are        further subdivided into several categories:    -   i. Distal axonopathies are the result of some metabolic or toxic        derangement of neurons. They may be caused by metabolic diseases        such as diabetes, renal failure, deficiency syndromes such as        malnutrition and alcoholism, or the effects of toxins or drugs.        Distal axonopathy (aka dying back neuropathy) is a type of        peripheral neuropathy that results from some metabolic or toxic        derangement of peripheral nervous system (PNS) neurons. It is        the most common response of nerves to metabolic or toxic        disturbances, and as such may be caused by metabolic diseases        such as diabetes, renal failure, deficiency syndromes such as        malnutrition and alcoholism, or the effects of toxins or drugs.        The most common cause of distal axonopathy is diabetes, and the        most common distal axonopathy is diabetic neuropathy.    -   ii. Myelinopathies are due to a primary attack on myelin causing        an acute failure of impulse conduction. The most common cause is        acute inflammatory demyelinating polyneuropathy (AIDP; aka        Guillain-Barré syndrome), though other causes include chronic        inflammatory demyelinating syndrome (CIDP), genetic metabolic        disorders (e.g., leukodystrophy), or toxins. Myelinopathy is due        to primary destruction of myelin or the myelinating Schwann        cells, which leaves the axon intact, but causes an acute failure        of impulse conduction. This demyelination slows down or        completely blocks the conduction of electrical impulses through        the nerve. The most common cause is acute inflammatory        demyelinating polyneuropathy (AIDP, better known as        Guillain-Barré syndrome), though other causes include chronic        inflammatory demyelinating polyneuropathy (CIDP), genetic        metabolic disorders (e.g., leukodystrophy or Charcot-Marie-Tooth        disease), or toxins.    -   iii. Neuronopathies are the result of destruction of peripheral        nervous system (PNS) neurons. They may be caused by motor        neurone diseases, sensory neuronopathies (e.g., Herpes zoster),        toxins or autonomic dysfunction. Neurotoxins may cause        neuronopathies, such as the chemotherapy agent vincristine.        Neuronopathy is dysfunction due to damage to neurons of the        peripheral nervous system (PNS), resulting in a peripheral        neuropathy. It may be caused by motor neurone diseases, sensory        neuronopathies (e.g., Herpes zoster), toxic substances or        autonomic dysfunction. A person with neuronopathy may present in        different ways, depending on the cause, the way it affects the        nerve cells, and the type of nerve cell that is most affected.    -   iv. Focal entrapment neuropathies (e.g., carpal tunnel        syndrome).

II. Inflammatory Pain

The compounds of the invention may be used to treat pain caused by orotherwise associated with any of the following inflammatory conditions

A. Arthritic Disorder

Arthritic disorders include, for example, a rheumatoid arthritis; ajuvenile rheumatoid arthritis; a systemic lupus erythematosus (SLE); agouty arthritis; a scleroderma; an osteoarthritis; a psoriaticarthritis; an ankylosing spondylitis; a Reiter's syndrome (reactivearthritis); an adult Still's disease; an arthritis from a viralinfection; an arthritis from a bacterial infection, such as, e.g., agonococcal arthritis and a non-gonococcal bacterial arthritis (septicarthritis); a Tertiary Lyme disease; a tuberculous arthritis; and anarthritis from a fungal infection, such as, e,g. a blastomycosis

B. Autoimmune Diseases

Autoimmune diseases include, for example, a Guillain-Barré syndrome, aHashimoto's thyroiditis, a pernicious anemia, an Addison's disease, atype I diabetes, a systemic lupus erythematosus, a dermatomyositis, aSjogren's syndrome, a lupus erythematosus, a multiple sclerosis, amyasthenia gravis, a Reiter's syndrome and a Grave's disease.

C. Connective Tissue Disorder

Connective tissue disorders include, for example, a spondyloarthritis adermatomyositis, and a fibromyalgia.

D. Injury

Inflammation caused by injury, including, for example, a crush,puncture, stretch of a tissue or joint, may cause chronic inflammatorypain.

E. Infection

Inflammation caused by infection, including, for example, a tuberculosisor an interstitial keratitis may cause chronic inflammatory pain.

F. Neuritis

Neuritis is an inflammatory process affecting a nerve or group ofnerves. Symptoms depend on the nerves involved, but may include pain,paresthesias, paresis, or hypesthesia (numbness).

-   -   Examples include:    -   a. Brachial neuritis    -   b. Retrobulbar neuropathy, an inflammatory process affecting the        part of the optic nerve lying immediately behind the eyeball.    -   c. Optic neuropathy, an inflammatory process affecting the optic        nerve causing sudden, reduced vision in the affected eye. The        cause of optic neuritis is unknown. The sudden inflammation of        the optic nerve (the nerve connecting the eye and the brain)        leads to swelling and destruction of the myelin sheath. The        inflammation may occasionally be the result of a viral        infection, or it may be caused by autoimmune diseases such as        multiple sclerosis. Risk factors are related to the possible        causes.    -   d. Vestibular neuritis, a viral infection causing an        inflammatory process affecting the vestibular nerve.

G. Joint Inflammation

Inflammation of the joint, such as that caused by bursitis ortendonitis, for example, may cause chronic inflammatory pain.

III. Headache Pain

The compounds of the invention may be used to treat pain caused by orotherwise associated with any of the following headache conditions. Aheadache (medically known as cephalgia) is a condition of mild to severepain in the head; sometimes neck or upper back pain may also beinterpreted as a headache. It may indicate an underlying local orsystemic disease or be a disorder in itself.

A. Muscular/Myogenic Headache

Muscular/myogenic headaches appear to involve the tightening or tensingof facial and neck muscles; they may radiate to the forehead. Tensionheadache is the most common form of myogenic headache.

A tension headache is a condition involving pain or discomfort in thehead, scalp, or neck, usually associated with muscle tightness in theseareas. Tension headaches result from the contraction of neck and scalpmuscles. One cause of this muscle contraction is a response to stress,depression or anxiety. Any activity that causes the head to be held inone position for a long time without moving can cause a headache. Suchactivities include typing or use of computers, fine work with the hands,and use of a microscope. Sleeping in a cold room or sleeping with theneck in an abnormal position may also trigger this type of headache. Atension-type headache, includes, without limitation, an episodic tensionheadache and a chronic tension headache.

B. Vascular Headache

The most common type of vascular headache is migraine. Other kinds ofvascular headaches include cluster headaches, which cause repeatedepisodes of intense pain, and headaches resulting from high bloodpressure

-   -   1. Migraine    -   A migraine is a heterogeneous disorder that generally involves        recurring headaches. Migraines are different from other        headaches because they occur with other symptoms, such as, e.g.,        nausea, vomiting, or sensitivity to light. In most people, a        throbbing pain is felt only on one side of the head. Clinical        features such as type of aura symptoms, presence of prodromes,        or associated symptoms such as vertigo, may be seen in subgroups        of patients with different underlying pathophysiological and        genetic mechanisms. A migraine headache, includes, without        limitation, a migraine without aura (common migraine), a        migraine with aura (classic migraine), a menstrual migraine, a        migraine equivalent (acephalic headache), a complicated        migraine, an abdominal migraine and a mixed tension migraine.    -   2. Cluster headache    -   Cluster headaches affect one side of the head (unilateral) and        may be associated with tearing of the eyes and nasal congestion.        They occurs in clusters, happening repeatedly every day at the        same time for several weeks and then remitting.

D. High Blood Pressure Headache

E. Traction and Inflammatory Headache

Traction and inflammatory headaches are usually symptoms of otherdisorders, ranging from stroke to sinus infection.

F. Hormone Headache

G. Rebound Headache

Rebound headaches, also known as medication overuse headaches, occurwhen medication is taken too frequently to relieve headache. Reboundheadaches frequently occur daily and can be very painful.

H. Chronic Sinusitis Headache

Sinusitis is inflammation, either bacterial, fungal, viral, allergic orautoimmune, of the paranasal sinuses. Chronic sinusitis is one of themost common complications of the common cold. Symptoms include: Nasalcongestion; facial pain; headache; fever; general malaise; thick greenor yellow discharge; feeling of facial ‘fullness’ worsening on bendingover. In a small number of cases, chronic maxillary sinusitis can alsobe brought on by the spreading of bacteria from a dental infection.Chronic hyperplastic eosinophilic sinusitis is a noninfective form ofchronic sinusitis.

I. An Organic Headache

J. Ictal Headaches

Ital headaches are headaches associated with seizure activity.

IV. Somatic Pain

The compounds of the invention may be used to treat pain caused by orotherwise associated with any of the following somatic pain conditions.Somatic pain originates from ligaments, tendons, bones, blood vessels,and even nerves themselves. It is detected with somatic nociceptors. Thescarcity of pain receptors in these areas produces a dull,poorly-localized pain of longer duration than cutaneous pain; examplesinclude sprains and broken bones. Additional examples include thefollowing.

A. Excessive Muscle Tension

Excessive muscle tension can be caused, for example, by a sprain or astrain.

B. Repetitive Motion Disorders

Repetitive motion disorders can result from overuse of the hands,wrists, elbows, shoulders, neck, back, hips, knees, feet, legs, orankles.

C. Muscle Disorders

Muscle disorders causing somatic pain include, for example, apolymyositis, a dermatomyositis, a lupus, a fibromyalgia, a polymyalgiarheumatica, and a rhabdomyolysis.

D. Myalgia

Myalgia is muscle pain and is a symptom of many diseases and disorders.The most common cause for myalgia is either overuse or over-stretchingof a muscle or group of muscles. Myalgia without a traumatic history isoften due to viral infections. Longer-term myalgias may be indicative ofa metabolic myopathy, some nutritional deficiencies or chronic fatiguesyndrome.

E. Infection

Infection can cause somatic pain. Examples of such infection include,for example, an abscess in the muscle, a trichinosis, an influenza, aLyme disease, a malaria, a Rocky Mountain spotted fever, Avianinfluenza, the common cold, community-acquired pneumonia, meningitis,monkeypox, Severe Acute Respiratory Syndrome, toxic shock syndrome,trichinosis, typhoid fever, and upper respiratory tract infection.

F. Drugs

Drugs can cause somatic pain. Such drugs include, for example, cocaine,a statin for lowering cholesterol (such as atorvastatin, simvastatin,and lovastatin), and an ACE inhibitor for lowering blood pressure (suchas enalapril and captopril)

V. Visceral Pain

The compounds of the invention may be used to treat pain caused by orotherwise associated with any of the following visceral pain conditions.Visceral pain originates from body's viscera, or organs. Visceralnociceptors are located within body organs and internal cavities. Theeven greater scarcity of nociceptors in these areas produces pain thatis usually more aching and of a longer duration than somatic pain.Visceral pain is extremely difficult to localise, and several injuriesto visceral tissue exhibit “referred” pain, where the sensation islocalised to an area completely unrelated to the site of injury.Examples of visceral pain include the following.

A. Functional Visceral Pain

Functional visceral pain includes, for example, an irritable bowelsyndrome and a chronic functional abdominal pain (CFAP), a functionalconstipation and a functional dyspepsia, a non-cardiac chest pain (NCCP)and a chronic abdominal pain.

B. Chronic Gastrointestinal Inflammation

Chronic gastrointestinal inflammation includes, for example, agastritis, an inflammatory bowel disease, like, e.g., a Crohn's disease,an ulcerative colitis, a microscopic colitis, a diverticulitis and agastroenteritis; an interstitial cystitis; an intestinal ischemia; acholecystitis; an appendicitis; a gastroesophageal reflux; an ulcer, anephrolithiasis, an urinary tract infection, a pancreatitis and ahernia.

C. Autoimmune Pain

Autoimmune pain includes, for example, a sarcoidosis and a vasculitis.

D. Organic Visceral Pain

Organic visceral pain includes, for example, pain resulting from atraumatic, inflammatory or degenerative lesion of the gut or produced bya tumor impinging on sensory innervation.

E. Treatment-Induced Visceral Pain

Treatment-induced visceral pain includes, for example, a pain attendantto chemotherapy therapy or a pain attendant to radiation therapy.

VI. Referred Pain

The compounds of the invention may be used to treat pain caused by orotherwise associated with any of the following referred pain conditions.

Referred pain arises from pain localized to an area separate from thesite of pain stimulation. Often, referred pain arises when a nerve iscompressed or damaged at or near its origin. In this circumstance, thesensation of pain will generally be felt in the territory that the nerveserves, even though the damage originates elsewhere. A common exampleoccurs in intervertebral disc herniation, in which a nerve root arisingfrom the spinal cord is compressed by adjacent disc material. Althoughpain may arise from the damaged disc itself, pain will also be felt inthe region served by the compressed nerve (for example, the thigh, knee,or foot). Relieving the pressure on the nerve root may ameliorate thereferred pain, provided that permanent nerve damage has not occurred.Myocardial ischaemia (the loss of blood flow to a part of the heartmuscle tissue) is possibly the best known example of referred pain; thesensation can occur in the upper chest as a restricted feeling, or as anache in the left shoulder, arm or even hand.

The present invention addresses a wide range of pain conditions, inparticular chronic pain conditions. Preferred conditions includecancerous and non-cancerous pain, inflammatory pain and neuropathicpain. The opioid-fusions of the present application are particularlysuited to addressing inflammatory pain, though may be less suited toaddressing neuropathic pain. The galanin-fusions are more suited toaddressing neuropathic pain.

In use, the polypeptides of the present invention are typically employedin the form of a pharmaceutical composition in association with apharmaceutical carrier, diluent and/or excipient, although the exactform of the composition may be tailored to the mode of administration.Administration is preferably to a mammal, more preferably to a human.

The polypeptides may, for example, be employed in the form of a sterilesolution for intra-articular administration or intra-cranialadministration. Spinal injection (e.g. epidural or intrathecal) ispreferred.

The dosage ranges for administration of the polypeptides of the presentinvention are those to produce the desired therapeutic effect. It willbe appreciated that the dosage range required depends on the precisenature of the components, the route of administration, the nature of theformulation, the age of the patient, the nature, extent or severity ofthe patient's condition, contraindications, if any, and the judgement ofthe attending physician.

Suitable daily dosages are in the range 0.0001-1 mg/kg, preferably0.0001-0.5 mg/kg, more preferably 0.002-0.5 mg/kg, and particularlypreferably 0.004-0.5 mg/kg. The unit dosage can vary from less that 1microgram to 30 mg, but typically will be in the region of 0.01 to 1 mgper dose, which may be administered daily or preferably less frequently,such as weekly or six monthly.

A particularly preferred dosing regimen is based on 2.5 ng of fusionprotein as the 1× dose. In this regard, preferred dosages are in therange 1×-100× (i.e. 2.5-250 ng). This dosage range is significantlylower (i.e. at least 10-fold, typically 100-fold lower) than would beemployed with other types of analgesic molecules such as NSAIDS,morphine, and gabapentin. Moreover, the above-mentioned difference isconsiderably magnified when the same comparison is made on a molarbasis—this is because the fusion proteins of the present invention havea considerably greater Mw than do conventional ‘small’ moleculetherapeutics.

Wide variations in the required dosage, however, are to be expecteddepending on the precise nature of the components, and the differingefficiencies of various routes of administration.

Variations in these dosage levels can be adjusted using standardempirical routines for optimisation, as is well understood in the art.

Compositions suitable for injection may be in the form of solutions,suspensions or emulsions, or dry powders which are dissolved orsuspended in a suitable vehicle prior to use.

Fluid unit dosage forms are typically prepared utilising a pyrogen-freesterile vehicle. The active ingredients, depending on the vehicle andconcentration used, can be either dissolved or suspended in the vehicle.

In preparing administrable solutions, the polypeptides can be dissolvedin a vehicle, the solution being made isotonic if necessary by additionof sodium chloride and sterilised by filtration through a sterile filterusing aseptic techniques before filling into suitable sterile vials orampoules and sealing. Alternatively, if solution stability is adequate,the solution in its sealed containers may be sterilised by autoclaving.

Advantageously additives such as buffering, solubilising, stabilising,preservative or bactericidal, suspending or emulsifying agents may bedissolved in the vehicle.

Dry powders which are dissolved or suspended in a suitable vehicle priorto use may be prepared by filling pre-sterilised drug substance andother ingredients into a sterile container using aseptic technique in asterile area.

Alternatively the polypeptides and other ingredients may be dissolved inan aqueous vehicle, the solution is sterilized by filtration anddistributed into suitable containers using aseptic technique in asterile area. The product is then freeze dried and the containers aresealed aseptically.

Parenteral suspensions, suitable for intramuscular, subcutaneous orintradermal injection, are prepared in substantially the same manner,except that the sterile components are suspended in the sterile vehicle,instead of being dissolved and sterilisation cannot be accomplished byfiltration. The components may be isolated in a sterile state oralternatively it may be sterilised after isolation, e.g. by gammairradiation.

Advantageously, a suspending agent for example polyvinylpyrrolidone isincluded in the composition/s to facilitate uniform distribution of thecomponents.

Definitions Section

Targeting Moiety (TM) means any chemical structure associated with anagent that functionally interacts with a Binding Site to cause aphysical association between the agent and the surface of a target cell.In the context of the present invention, the target cell is anociceptive sensory afferent. The term TM embraces any molecule (i.e. anaturally occurring molecule, or a chemically/physically modifiedvariant thereof) that is capable of binding to a Binding Site on thetarget cell, which Binding Site is capable of internalisation (e.g.endosome formation)—also referred to as receptor-mediated endocytosis.The TM may possess an endosomal membrane translocation function, inwhich case separate TM and Translocation Domain components need not bepresent in an agent of the present invention.

The TM of the present invention binds (preferably specifically binds) toa nociceptive sensory afferent (e.g. a primary nociceptive afferent). Inthis regard, specifically binds means that the TM binds to a nociceptivesensory afferent (e.g. a primary nociceptive afferent) with a greateraffinity than it binds to other neurons such as non-nociceptiveafferents, and/or to motor neurons (i.e. the natural target forclostridial neurotoxin holotoxin). The term “specifically binding” canalso mean that a given TM binds to a given receptor, for example galaninreceptors, such as GALR1, GALR2 and/or GALR3 receptors, with a bindingaffinity (Ka) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ or greater, morepreferably 10⁸ M⁻¹ or greater, and most preferably, 10⁹ M⁻¹ or greater.

For the purposes of this invention, an agonist is defined as a moleculethat is capable of stimulating the process of exocytic fusion in atarget cell, which process is susceptible to inhibition by a proteasecapable of cleaving a protein of the exocytic fusion apparatus in saidtarget cell.

Accordingly, the particular agonist definition of the present inventionwould exclude many molecules that would be conventionally considered asagonists.

For example, nerve growth factor (NGF) is an agonist in respect of itsability to promote neuronal differentiation via binding to a TrkAreceptor. However, NGF is not an agonist when assessed by the abovecriteria because it is not a principal inducer of exocytic fusion. Inaddition, the process that NGF stimulates (i.e. cell differentiation) isnot susceptible to inhibition by the protease activity of anon-cytotoxic toxin molecule.

The term “fragment”, when used in relation to a protein, means a peptidehaving at least thirty-five, preferably at least twenty-five, morepreferably at least twenty, and most preferably at least 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 amino acid residues of theprotein in question.

The term “variant”, when used in relation to a protein, means a peptideor peptide fragment of the protein that contains one or more analoguesof an amino acid (e.g. an unnatural amino acid), or a substitutedlinkage.

The term “derivative”, when used in relation to a protein, means aprotein that comprises the protein in question, and a further peptidesequence. The further peptide sequence should preferably not interferewith the basic folding and thus conformational structure of the originalprotein. Two or more peptides (or fragments, or variants) may be joinedtogether to form a derivative. Alternatively, a peptide (or fragment, orvariant) may be joined to an unrelated molecule (e.g. a second,unrelated peptide). Derivatives may be chemically synthesized, but willbe typically prepared by recombinant nucleic acid methods. Additionalcomponents such as lipid, and/or polysaccharide, and/or polypeptidecomponents may be included.

The term non-cytotoxic means that the protease molecule in question doesnot kill the target cell to which it has been re-targeted.

The protease of the present invention embraces all naturally-occurringnon-cytotoxic proteases that are capable of cleaving one or moreproteins of the exocytic fusion apparatus in eukaryotic cells.

The non-cytotoxic protease of the present invention is preferably abacterial protease. In one embodiment, the non-cytotoxic protease isselected from the genera Clostridium or Neisseria (e.g. a clostridialL-chain, or a neisserial IgA protease preferably from N. gonorrhoeae).The term protease embraces functionally equivalent fragments andmolecules thereof.

The present invention also embraces modified non-cytotoxic proteases,which include amino acid sequences that do not occur in nature and/orsynthetic amino acid residues, so long as the modified proteases stilldemonstrate the above-mentioned protease activity.

The protease of the present invention preferably demonstrates a serineor metalloprotease activity (e.g. endopeptidase activity). The proteaseis preferably specific for a SNARE protein (e.g. SNAP-25,synaptobrevin/VAMP, or syntaxin).

Particular mention is made to the protease domains of neurotoxins, forexample the protease domains of bacterial neurotoxins. Thus, the presentinvention embraces the use of neurotoxin domains, which occur in nature,as well as recombinantly prepared versions of said naturally-occurringneurotoxins.

Exemplary neurotoxins are produced by clostridia, and the termclostridial neurotoxin embraces neurotoxins produced by C. tetani(TeNT), and by C. botulinum (BoNT) serotypes A-G, as well as the closelyrelated BoNT-like neurotoxins produced by C. baratii and C. butyricum.The above-mentioned abbreviations are used throughout the presentspecification. For example, the nomenclature BoNT/A denotes the sourceof neurotoxin as BoNT (serotype A). Corresponding nomenclature appliesto other BoNT serotypes.

The term L-chain or LC fragment means a component of the L-chain of aneurotoxin, which fragment demonstrates a metalloprotease activity andis capable of proteolytically cleaving a vesicle and/or plasma membraneassociated protein involved in cellular exocytosis.

A Translocation Domain is a molecule that enables translocation of aprotease (or fragment thereof) into a target cell such that a functionalexpression of protease activity occurs within the cytosol of the targetcell. Whether any molecule (e.g. a protein or peptide) possesses therequisite translocation function of the present invention may beconfirmed by any one of a number of conventional assays.

For example, Shone C. (1987) describes an in vitro assay employingliposomes, which are challenged with a test molecule. Presence of therequisite translocation function is confirmed by release from theliposomes of K⁺ and/or labelled NAD, which may be readily monitored [seeShone C. (1987) Eur. J. Biochem; vol. 167(1): pp. 175-180].

A further example is provided by Blaustein R. (1987), which describes asimple in vitro assay employing planar phospholipid bilayer membranes.The membranes are challenged with a test molecule and the requisitetranslocation function is confirmed by an increase in conductance acrosssaid membranes [see Blaustein (1987) FEBS Letts; vol. 226, no. 1: pp.115-120].

Additional methodology to enable assessment of membrane fusion and thusidentification of Translocation Domains suitable for use in the presentinvention are provided by Methods in Enzymology Vol 220 and 221,Membrane Fusion Techniques, Parts A and B, Academic Press 1993.

The Translocation Domain is preferably capable of formation ofion-permeable pores in lipid membranes under conditions of low pH.Preferably it has been found to use only those portions of the proteinmolecule capable of pore-formation within the endosomal membrane.

The Translocation Domain may be obtained from a microbial proteinsource, in particular from a bacterial or viral protein source. Hence,in one embodiment, the Translocation Domain is a translocating domain ofan enzyme, such as a bacterial toxin or viral protein.

It is well documented that certain domains of bacterial toxin moleculesare capable of forming such pores. It is also known that certaintranslocation domains of virally expressed membrane fusion proteins arecapable of forming such pores. Such domains may be employed in thepresent invention.

The Translocation Domain may be of a clostridial origin, namely theH_(N) domain (or a functional component thereof). H_(N) means a portionor fragment of the H-chain of a clostridial neurotoxin approximatelyequivalent to the amino-terminal half of the H-chain, or the domaincorresponding to that fragment in the intact H-chain. It is preferredthat the H-chain substantially lacks the natural binding function of theH_(C) component of the H-chain. In this regard, the H_(C) function maybe removed by deletion of the H_(C) amino acid sequence (either at theDNA synthesis level, or at the post-synthesis level by nuclease orprotease treatment). Alternatively, the H_(C) function may beinactivated by chemical or biological treatment. Thus, the H-chain ispreferably incapable of binding to the Binding Site on a target cell towhich native clostridial neurotoxin (i.e. holotoxin) binds.

In one embodiment, the translocation domain is a H_(N) domain (or afragment thereof) of a clostridial neurotoxin. Examples of suitableclostridial Translocation Domains include:

-   -   Botulinum type A neurotoxin—amino acid residues (449-871)    -   Botulinum type B neurotoxin—amino acid residues (441-858)    -   Botulinum type C neurotoxin—amino acid residues (442-866)    -   Botulinum type D neurotoxin—amino acid residues (446-862)    -   Botulinum type E neurotoxin—amino acid residues (423-845)    -   Botulinum type F neurotoxin—amino acid residues (440-864)    -   Botulinum type G neurotoxin—amino acid residues (442-863)    -   Tetanus neurotoxin—amino acid residues (458-879)

For further details on the genetic basis of toxin production inClostridium botulinum and C. tetani, we refer to Henderson et al (1997)in The Clostridia: Molecular Biology and Pathogenesis, Academic press.

The term H_(N) embraces naturally-occurring neurotoxin H_(N) portions,and modified H_(N) portions having amino acid sequences that do notoccur in nature and/or synthetic amino acid residues, so long as themodified H_(N) portions still demonstrate the above-mentionedtranslocation function.

Alternatively, the Translocation Domain may be of a non-clostridialorigin (see Table 4). Examples of non-clostridial Translocation Domainorigins include, but not be restricted to, the translocation domain ofdiphtheria toxin [O=Keefe et al., Proc. Natl. Acad. Sci. USA (1992) 89,6202-6206; Silverman et al., J. Biol. Chem. (1993) 269, 22524-22532; andLondon, E. (1992) Biochem. Biophys. Acta., 1112, pp. 25-51], thetranslocation domain of Pseudomonas exotoxin type A [Prior et al.Biochemistry (1992) 31, 3555-3559], the translocation domains of anthraxtoxin [Blanke et al. Proc. Natl. Acad. Sci. USA (1996) 93, 8437-8442], avariety of fusogenic or hydrophobic peptides of translocating function[Plank et al. J. Biol. Chem. (1994) 269, 12918-12924; and Wagner et al(1992) PNAS, 89, pp. 7934-7938], and amphiphilic peptides [Murata et al(1992) Biochem., 31, pp. 1986-1992]. The Translocation Domain may mirrorthe Translocation Domain present in a naturally-occurring protein, ormay include amino acid variations so long as the variations do notdestroy the translocating ability of the Translocation Domain.

Particular examples of viral Translocation Domains suitable for use inthe present invention include certain translocating domains of virallyexpressed membrane fusion proteins. For example, Wagner et al. (1992)and Murata et al. (1992) describe the translocation (i.e. membranefusion and vesiculation) function of a number of fusogenic andamphiphilic peptides derived from the N-terminal region of influenzavirus haemagglutinin. Other virally expressed membrane fusion proteinsknown to have the desired translocating activity are a translocatingdomain of a fusogenic peptide of Semliki Forest Virus (SFV), atranslocating domain of vesicular stomatitis virus (VSV) glycoprotein G,a translocating domain of SER virus F protein and a translocating domainof Foamy virus envelope glycoprotein. Virally encoded Aspike proteinshave particular application in the context of the present invention, forexample, the E1 protein of SFV and the G protein of the G protein ofVSV.

Use of the Translocation Domains listed in Table (below) includes use ofsequence variants thereof. A variant may comprise one or moreconservative nucleic acid substitutions and/or nucleic acid deletions orinsertions, with the proviso that the variant possesses the requisitetranslocating function. A variant may also comprise one or more aminoacid substitutions and/or amino acid deletions or insertions, so long asthe variant possesses the requisite translocating function.

Translocation Amino acid domain source residues References Diphtheriatoxin 194-380 Silverman et al., 1994, J. Biol. Chem. 269, 22524-22532London E., 1992, Biochem. Biophys. Acta., 1113, 25-51 Domain II of405-613 Prior et al., 1992, Biochemistry pseudomonas 31, 3555-3559exotoxin Kihara & Pastan, 1994, Bioconj Chem. 5, 532-538 Influenza virusGLFGAIAGFIENGWE Plank et al., 1994, J. Biol. Chem. haemagglutininGMIDGWYG, and 269, 12918-12924 Variants thereof Wagner et al., 1992,PNAS, 89, 7934-7938 Murata et al., 1992, Biochemistry 31, 1986-1992Semliki Forest virus Translocation domain Kielian et al., 1996, J CellBiol. fusogenic protein 134(4), 863-872 Vesicular Stomatitis 118-139 Yaoet al., 2003, Virology 310(2), virus glycoprotein G 319-332 SER virus Fprotein Translocation domain Seth et al., 2003, J Virol 77(11) 6520-6527Foamy virus envelope Translocation domain Picard-Maureau et al., 2003, Jglycoprotein Virol. 77(8), 4722-4730

There now follows a brief description of the Figures, which illustrateaspects and/or embodiments of the present invention.

FIG. 1—Purification of a LC/A-spacer-galanin-spacer-H_(N)/A fusionprotein

Using the methodology outlined in Example 3, aLC/A-GS18-galanin-GS20-H_(N)/A fusion protein was purified from E. coliBL21 cells. Briefly, the soluble products obtained following celldisruption were applied to a nickel-charged affinity capture column.Bound proteins were eluted with 100 mM imidazole, treated withenterokinase to activate the fusion protein and treated with factor Xato remove the maltose-binding protein (MBP) tag. Activated fusionprotein was then re-applied to a second nickel-charged affinity capturecolumn. Samples from the purification procedure were assessed bySDS-PAGE (Panel A) and Western blotting (Panel B). Anti-galanin antisera(obtained from Abcam) and Anti-histag antisera (obtained from Qiagen)were used as the primary antibody for Western blotting. The finalpurified material in the absence and presence of reducing agent isidentified in the lanes of Panel A marked [−] and [+] respectively.Panel A, Lane 1=Benchmark ladder; 2=soluble fraction; 3=1^(st) Hisproduct; 4=activated purified protein; 5=second His product; 6=finalpurified protein 5 μl; 7=final purified protein 10 μl; 8=final purifiedprotein 20 μl; 9=final purified protein 5 μl+DTT; 10=final purifiedprotein 10 μl+DTT. Panel B Lane 1=Benchmark ladder; 2=soluble fraction;3=1^(st) His product; 4=activated purified protein; 5=second Hisproduct; 6=final purified protein 2 μl; 7=final purified protein 5 μl;8=final purified protein 10 μl; 9=final purified protein 2 μl+DTT;10=final purified protein 5 μl+DTT.

FIG. 2—Purification of a LC/C-spacer-galanin-spacer-H_(N)/C fusionprotein

Using the methodology outlined in Example 3, an LC/C-galanin-H_(N)/Cfusion protein was purified from E. coli BL21 cells. Briefly, thesoluble products obtained following cell disruption were applied to anickel-charged affinity capture column. Bound proteins were eluted with100 mM imidazole, treated with enterokinase to activate the fusionprotein, then re-applied to a second nickel-charged affinity capturecolumn. Samples from the purification procedure were assessed bySDS-PAGE (Panel A) and Western blotting (Panel B). Anti-galanin antisera(obtained from Abcam) and Anti-histag antisera (obtained from Qiagen)were used as the primary antibody for Western blotting. The finalpurified material in the absence and presence of reducing agent in PanelA is identified in the lanes marked [−] and [+] respectively. Panel A,Lane 1=Benchmark ladder; 2=soluble fraction; 3=product 1^(st) column;4=enterokinase activated protein; 5=final product 0.1 mg/ml (5 μl);6=final product 0.1 mg/ml+DTT (5 μl); 7=final product 0.1 mg/ml (10 μl);8=final product 0.1 mg/ml+DTT (10 μl). Panel B, Lane 1=Magic mark;2=soluble fraction; 3=product 1^(st) His-tag column; 4=activated fusion;5=purified @ 0.1 mg/ml (5 μl); 6=purified @ 0.1 mg/ml+DTT (5 μl); 7purified @ 0.1 mg/ml+100 mm DTT (10 μl); 8=purified @ 0.1 mg/ml+100 mmDTT (10 μl)+DTT.

FIG. 3—Comparison of SNARE cleavage efficacy of aLC-spacer-galanin-spacer-H_(N) fusion protein and a LC-H_(N)-galaninfusion protein

Panels A & B: The ability of galanin fusions to cleave SNAP-25 in a CHOGALR1 SNAP25 cells was assessed. Chinese hamster ovary (CHO) cells weretransfected so that they express the GALR1 receptor. Said cells werefurther transfected to express a SNARE protein (SNAP-25). Thetransfected cells were exposed to varying concentrations of differentgalanin fusion proteins for 24 hours. Cellular proteins were separatedby SDS-PAGE, Western blotted, and probed with anti-SNAP-25 to facilitatean assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 wascalculated by densitometric analysis. It is clear from the data that theLC-spacer-galanin-spacer-H_(N) fusion (Fusion 1) is more potent than theLC-H_(N)-galanin fusion and the unliganded LC/A-H_(N)/A controlmolecule.

FIG. 4—GALR1 receptor activation studies in the CHO-GALCHO-GALR1 SNAP-25cleavage assay with galanin fusion proteins of the present inventionhaving different serotype backbones

Chinese hamster ovary (CHO) cells were transfected so that they expressthe GALR1 receptor and SNAP-25. Said cells were used to measure cAMPdeletion that occurs when the receptor is activated with a galaninligand, using a FRET-based cAMP kit (LANCE kit from Perkin Elmer). Thetransfected cells were exposed to varying concentrations of galanin(GA16) fusion proteins having different serotype backbones (i.e.botulinum neurotoxin serotypes A, B, C and D) for 2 hours. cAMP levelswere then detected by addition of a detection mix containing afluorescently labelled cAMP tracer (Europium-streptavadi/biotin-cAMP)and fluorescently (Alexa) labelled anti-cAMP antibody and incubating atroom temperature for 24 hours. Then samples are excited at 320 nM andemitted light measured at 665 nM to determine cAMP levels. The datademonstrate that galanin fusion proteins of the present invention havingdifferent serotype backbones activated the GALR1 receptor.

FIG. 5—Cleavage of SNARE protein by galanin (GA16 and GA30) fusionproteins in CHO-GALR1 SNAP-25 cleavage assay

Chinese hamster ovary (CHO) cells were transfected so that they expressthe GALR1 receptor. Said cells were further transfected to express aSNARE protein (SNAP-25). The transfected cells were exposed to varyingconcentrations of different galanin fusion proteins for 24 hours.Cellular proteins were separated by SDS-PAGE, Western blotted, andprobed with anti-SNAP-25 to facilitate an assessment of SNAP-25cleavage. The percentage of cleaved SNAP-25 was calculated bydensitometric analysis. The data demonstrate that galanin fusionproteins having galanin-16 and galanin-30 ligands cleave SNARE protein.In addition, the data confirm that galanin fusion proteins having GS5,GS10 and GS18 spacers between the non-cytotoxic protease component andthe protease cleavage site are functional.

FIG. 6—Results of in vivo paw guarding assay employing galanin fusionproteins

The nociceptive flexion reflex (also known as paw guarding assay) is arapid withdrawal movement that constitutes a protective mechanismagainst possible limb damage. It can be quantified by assessment ofelectromyography (EMG) response in anesthetized rat as a result of lowdose capsaicin, electrical stimulation or the capsaicin-sensitizedelectrical response. Intraplantar pretreatment (24 hour) of fusionproteins of the present invention into 300-380 g male Sprague-Dawleyrats. Induction of paw guarding was achieved by 0.006% capsaicin, 10 μlin PBS (7.5% DMSO), injected in 10 seconds. This produced a robustreflex response from biceps feroris muscle. A reduction/inhibition ofthe nociceptive flexion reflex indicates that the test substancedemonstrates an antinociceptive effect. The data demonstrated theantinociceptive effect of the galanin fusion proteins of the presentinvention.

FIG. 7—Galanin fusion protein efficacy in capsaicin-induced thermalhyperalgesia assay

The ability of different galanin fusion proteins of the invention toinhibit capsaicin-induced thermal hyperalgesia was evaluated.Intraplantar pretreatment of fusion proteins into Sprague-Dawley ratsand 24 hours later 0.3% capsaicin was injected and rats were put on 25°C. glass plate (rats contained in acrylic boxes, on 25° C. glass plate).Light beam (adjustable light Intensity) focused on the hind paw. Sensorsdetected movement of paw, stopping timer. Paw Withdrawal Latency is timeto remove paw from heat source (Cut-off of 20.48 seconds). Areduction/inhibition of the paw withdrawal latency indicates that thetest substance demonstrates an antinociceptive effect. No.1=LC_H_(N)-GA16; No. 2=LC-H_(N)-GA30; No.3=LC-GS5-EN-CPGA16-GS20-H_(N)-HT; No. 4=LC-GS18-EN-CPGA16-GS20-H_(N)-HT;No. 5=BOTOX; No. 6=morphine. The data demonstrated the enhancedantinociceptive effect of the galanin fusion proteins of the presentinvention compared to fusion proteins with a C-terminally presentedligand.

FIG. 8—Galanin fusion protein efficacy in capsaicin-induced thermalhyperalgesia assay

The ability of different galanin fusion proteins of the invention toinhibit capsaicin-induced thermal hyperalgesia was evaluated.Intraplantar pretreatment of fusion proteins into Sprague-Dawley ratsand 24 hours later 0.3% capsaicin was injected and rats were put on 25°C. glass plate (rats contained in acrylic boxes, on 25° C. glass plate).Light beam (adjustable light Intensity) focused on the hind paw. Sensorsdetected movement of paw, stopping timer. Paw Withdrawal Latency is timeto remove paw from heat source (Cut-off of 20.48 seconds). Areduction/inhibition of the paw withdrawal latency indicates that thetest substance demonstrates an antinociceptive effect. The datademonstrated the antinociceptive effect of the galanin fusion proteinsof the present invention having different serotype backbones (i.e. A, B,C and D).

FIG. 9—Activation of galanin fusion proteins with single anddouble-spacers

Galanin fusion proteins lacking a first spacer (spacer 1) of the presentinvention located between the non-cytotoxic protease component and theTargeting Moiety component showed poor activation with protease (PanelsA and B). Panel C demonstrates the enhanced activation of galanin fusionproteins of the present invention having both first (spacer 1) andsecond (spacer 2) spacers. Panels A&B: 1) Benchmark ladder; 2)Unactivated control; 3) Unactivated control+DTT; 4) Protease activatedprotein+0.0 mM ZnCl2; 5) Protease activated protein+0.0 mM ZnCl2+DTT; 6)Protease activated protein+0.2 mM ZnCl2; 7) Protease activatedprotein+0.2 mM ZnCl2+DTT; 8) Protease activated protein+0.4 mM ZnCl2; 9)Protease activated protein+0.4 mM ZnCl2+DTT; 10) Protease activatedprotein+0.8 mM ZnCl2; 11) Protease activated protein+0.8 mM ZnCl2+DTT.Panel C: 1) Benchmark ladder; 2) Unactivated control 25° C.; 3)Unactivated control 25° C.+DTT; 4) Protease activated protein 25° C.; 5)Protease activated protein 25° C.+DTT; 6) Benchmark ladder.

SEQ ID NOS

Where an initial Met amino acid residue or a corresponding initial codonis indicated in any of the following SEQ ID NOs, said residue/codon isoptional.

SEQ ID NO 1 DNA sequence of the LC/A SEQ ID NO 2 DNA sequence of theH_(N)/A SEQ ID NO 3 DNA sequence of the LC/B SEQ ID NO 4 DNA sequence ofthe H_(N)/B SEQ ID NO 5 DNA sequence of the LC/C SEQ ID NO 6 DNAsequence of the H_(N)/C SEQ ID NO 7 Protein sequence of galanin GA30 SEQID NO 8 Protein sequence of galanin GA16 SEQ ID NO 9 DNA sequence ofLC/A-GS18-EN-CPGA16-GS20-H_(N)/A-HT SEQ ID NO 10 Protein sequence ofLC/A-GS18-EN-CPGA16-GS20-H_(N)/A-HT SEQ ID NO 11 Protein sequence ofLC/A-GS18-EN-CPGA16-GS20-H_(N)/A SEQ ID NO 12 DNA sequence ofLC/A-GS5-EN-CPGA16-GS20-H_(N)/A-HT SEQ ID NO 13 Protein sequence ofLC/A-GS5-EN-CPGA16-GS20-H_(N)/A-HT SEQ ID NO 14 Protein sequence ofLC/A-GS5-EN-CPGA16-H_(N)/A-GS20 SEQ ID NO 15 DNA sequence ofLC/A-GS5-EN-CPGA30-GS20-H_(N)/A-HT SEQ ID NO 16 Protein sequence ofLC/A-GS5-EN-CPGA30-GS20-H_(N)/A-HT SEQ ID NO 17 Protein sequence ofLC/A-GS5-EN-CPGA30-GS20-H_(N)/A SEQ ID NO 18 DNA sequence ofLC/B-GS5-EN-CPGA16-GS20-H_(N)/B(K191A)-HT SEQ ID NO 19 Protein sequenceof LC/B-GS5-EN-CPGA16-GS20-H_(N)/B(K191A)-HT SEQ ID NO 20 Proteinsequence of LC/B-GS5-EN-CPGA16-GS20-H_(N)/B(K191A) SEQ ID NO 21 DNAsequence of LC/B-GS5-EN-CPGA16-GS20-H_(N)/B-HT SEQ ID NO 22 Proteinsequence of LC/B-GS5-EN-CPGA16-GS20-H_(N)/B-HT SEQ ID NO 23 Proteinsequence of LC/B-GS5-EN-CPGA16-GS20-H_(N)/B SEQ ID NO 24 DNA sequence ofLC/C-GS5-EN-CPGA16-GS20-H_(N)/C-HT SEQ ID NO 25 Protein sequence ofLC/C-GS5-EN-CPGA16-GS20-H_(N)/C-HT SEQ ID NO 26 Protein sequence ofLC/C-GS5-EN-CPGA16-GS20-H_(N)/C SEQ ID NO 27 DNA sequence ofLC/D-GS5-EN-CPGA16-GS20-H_(N)/D-HT SEQ ID NO 28 Protein sequence ofLC/D-GS5-EN-CPGA16-GS20-H_(N)/D-HT SEQ ID NO 29 Protein sequence ofLC/D-GS5-EN-CPGA16-H_(N)/D-GS20 SEQ ID NO 30 DNA sequence ofLC/A-GS5-EN-CPGA16-HX27-H_(N)/A-HT SEQ ID NO 31 Protein sequence ofLC/A-GS5-EN-CPGA16-HX27-H_(N)/A-HT SEQ ID NO 32 Protein sequence ofLC/A-GS5-EN-CPGA16-HX27-H_(N)/A- SEQ ID NO 33 Protein sequence ofLC/A-GS10-EN-CPGA16-H_(N)/A-GS20-HT SEQ ID NO 34 Protein sequence ofLC/A-GS10-EN-CPGA16-GS20-H_(N)/A SEQ ID NO 35 Protein sequence ofLC/A-GS5-EN-CPGA16-GS15-H_(N)/A-HT SEQ ID NO 36 Protein sequence ofLC/A-GS5-EN-CPGA16-GS15-H_(N)/A SEQ ID NO 37 Protein sequence ofLC/A-GS5-EN-CPGA16-GS10-H_(N)/A-HT SEQ ID NO 38 Protein sequence ofLC/A-GS5-EN-CPGA16-GS10-H_(N)/A SEQ ID NO 39 Protein sequence ofLC/A-GS18-EN-CPGA16-HX27-H_(N)/A-HT SEQ ID NO 40 Protein sequence ofLC/A-GS18-EN-CPGA16-HX27-H_(N)/A SEQ ID NO 41 Protein sequence ofLC/A-GS18-EN-CPGA16-GS15-H_(N)/A-HT SEQ ID NO 42 Protein sequence ofLC/A-GS18-EN-CPGA16-GS15 SEQ ID NO 43 Protein sequence ofLC/A-GS18-EN-CPGA16-GS10-HT SEQ ID NO 44 Protein sequence ofLC/A-GS18-EN-CPGA16-GS10 SEQ ID NO 45 Protein sequence ofLC/A-GS10-EN-CPGA16-HX27-HT SEQ ID NO 46 Protein sequence ofLC/A-GS10-EN-CPGA16-HX27 SEQ ID NO 47 Protein sequence ofLC/A-GS10-EN-CPGA16-GS15-H_(N)/A-HT SEQ ID NO 48 Protein sequence ofLC/A-GS10-EN-CPGA16-GS15-H_(N)/A SEQ ID NO 49 Protein sequence ofLC/A-GS10-EN-CPGA16-GS10-H_(N)/A-HT SEQ ID NO 50 Protein sequence ofLC/A-GS10-EN-CPGA16-GS10-H_(N)/A SEQ ID NO 51 DNA sequence of the IgAprotease SEQ ID NO 52 DNA sequence of the IgA-GS5-CPGA16-GS20-H_(N)/Afusion SEQ ID NO 53 Protein sequence of the IgA-GS5-CPGA16-GS20-H_(N)/Afusion SEQ ID NO 54 DNA sequence of DT translocation domain SEQ ID NO 55DNA sequence of LC/A-GS5-GA16-GS20-DT SEQ ID NO 56 Protein sequence ofLC/A-GS5-GA16-GS20-DT SEQ ID NO 57 DNA sequence of TeNT LC SEQ ID NO 58DNA sequence of TeNT LC-GS5--CPGA16-GS20-H_(N)/A SEQ ID NO 59 Proteinsequence of TeNT LC-GS5-EN-CPGA16-GS20-H_(N)/A

EXAMPLES Example 1—Construction and Activation of Galanin FusionProteins

Preparation of a LC/A and H_(N)/A Backbone Clones

The following procedure creates the LC and H_(N) fragments for use asthe component backbone for multidomain fusion expression. This exampleis based on preparation of a serotype A based clone (SEQ ID NO1 and SEQID NO2), though the procedures and methods are equally applicable to theother serotypes (i.e. A, B, C, D and E serotypes) as illustrated by thesequence listing for serotype B (SEQ ID NO3 and SEQ ID NO4) and serotypeC (SEQ ID NO5 and SEQ ID NO6)].

Preparation of Cloning and Expression Vectors

pCR 4 (Invitrogen) is the chosen standard cloning vector, selected dueto the lack of restriction sequences within the vector and adjacentsequencing primer sites for easy construct confirmation. The expressionvector is based on the pMAL (NEB) expression vector, which has thedesired restriction sequences within the multiple cloning site in thecorrect orientation for construct insertion (BamHI-SalI-PstI-HindIII). Afragment of the expression vector has been removed to create anon-mobilisable plasmid and a variety of different fusion tags have beeninserted to increase purification options.

Preparation of Protease (e.g. LC/A) Insert

The LC/A (SEQ ID NO1) is created by one of two ways:

The DNA sequence is designed by back translation of the LC/A amino acidsequence [obtained from freely available database sources such asGenBank (accession number P10845) or Swissprot (accession locusBXA1_CLOBO) using one of a variety of reverse translation software tools(for example EditSeq best E. coli reverse translation (DNASTAR Inc.), orBacktranslation tool v2.0 (Entelechon)]. BamHI/SalI recognitionsequences are incorporated at the 5′ and 3′ ends respectively of thesequence, maintaining the correct reading frame. The DNA sequence isscreened (using software such as MapDraw, DNASTAR Inc.) for restrictionenzyme cleavage sequences incorporated during the back translation. Anycleavage sequences that are found to be common to those required by thecloning system are removed manually from the proposed coding sequenceensuring common E. coli codon usage is maintained. E. coli codon usageis assessed by reference to software programs such as Graphical CodonUsage Analyser (Geneart), and the % GC content and codon usage ratioassessed by reference to published codon usage tables (for exampleGenBank Release 143, 13 Sep. 2004). This optimised DNA sequencecontaining the LC/A open reading frame (ORF) is then commerciallysynthesized (for example by Entelechon, Geneart or Sigma-Genosys) and isprovided in the pCR 4 vector.

The alternative method is to use PCR amplification from an existing DNAsequence with BamHI and SalI restriction enzyme sequences incorporatedinto the 5′ and 3′ PCR primers respectively. Complementaryoligonucleotide primers are chemically synthesised by a supplier (forexample MWG or Sigma-Genosys), so that each pair has the ability tohybridize to the opposite strands (3′ ends pointing “towards” eachother) flanking the stretch of Clostridium target DNA, oneoligonucleotide for each of the two DNA strands. To generate a PCRproduct the pair of short oligonucleotide primers specific for theClostridium DNA sequence are mixed with the Clostridium DNA template andother reaction components and placed in a machine (the ‘PCR machine’)that can change the incubation temperature of the reaction tubeautomatically, cycling between approximately 94° C. (for denaturation),55° C. (for oligonucleotide annealing), and 72° C. (for synthesis).Other reagents required for amplification of a PCR product include a DNApolymerase (such as Taq or Pfu polymerase), each of the four nucleotidedNTP building blocks of DNA in equimolar amounts (50-200 μM) and abuffer appropriate for the enzyme optimised for Mg²⁺ concentration(0.5-5 mM).

The amplification product is cloned into pCR 4 using either, TOPO TAcloning for Taq PCR products or Zero Blunt TOPO cloning for Pfu PCRproducts (both kits commercially available from Invitrogen). Theresultant clone is checked by sequencing. Any additional restrictionsequences which are not compatible with the cloning system are thenremoved using site directed mutagenesis [for example, using Quickchange(Stratagene Inc.)].

Preparation of Translocation (e.g. H_(N)) Insert

The H_(N)/A (SEQ ID NO2) is created by one of two ways:

The DNA sequence is designed by back translation of the H_(N)/A aminoacid sequence [obtained from freely available database sources such asGenBank (accession number P10845) or Swissprot (accession locusBXA1_CLOBO)] using one of a variety of reverse translation softwaretools [for example EditSeq best E. coli reverse translation (DNASTARInc.), or Backtranslation tool v2.0 (Entelechon)]. A PstI restrictionsequence added to the N-terminus and XbaI-stop codon-HindIII to theC-terminus ensuring the correct reading frame is maintained. The DNAsequence is screened (using software such as MapDraw, DNASTAR Inc.) forrestriction enzyme cleavage sequences incorporated during the backtranslation. Any sequences that are found to be common to those requiredby the cloning system are removed manually from the proposed codingsequence ensuring common E. coli codon usage is maintained. E. colicodon usage is assessed by reference to software programs such asGraphical Codon Usage Analyser (Geneart), and the % GC content and codonusage ratio assessed by reference to published codon usage tables (forexample GenBank Release 143, 13 Sep. 2004). This optimised DNA sequenceis then commercially synthesized (for example by Entelechon, Geneart orSigma-Genosys) and is provided in the pCR 4 vector.

The alternative method is to use PCR amplification from an existing DNAsequence with PstI and XbaI-stop codon-HindIII restriction enzymesequences incorporated into the 5′ and 3′ PCR primers respectively. ThePCR amplification is performed as described above. The PCR product isinserted into pCR 4 vector and checked by sequencing. Any additionalrestriction sequences which are not compatible with the cloning systemare then removed using site directed mutagenesis [for example usingQuickchange (Stratagene Inc.)].

Preparation of LC/A-GS18-EN-CPGA16-GS20-H_(N)/A Fusion

In order to create the LC/A-GS18-EN-CPGA16-GS20-H_(N)/A construct, an Aserotype linker with the addition of an Enterokinase site foractivation, arranged as BamHI-SalI-GS18-proteasesite-GS20-PstI-XbaI-stop codon-HindIII is synthesised. The pCR 4 vectorencoding the linker is cleaved with BamHI+SalI restriction enzymes. Thiscleaved vector then serves as the recipient for insertion and ligationof the LC/A DNA (SEQ ID NO1) also cleaved with BamHI+SalI. Thisconstruct is then cleaved with BamHI+HindIII and inserted into anexpression vector such as the pMAL plasmid (NEB) or pET based plasmid(Novagen). The resulting plasmid DNA is then cleaved with PstI+XbaIrestriction enzymes and the H_(N)/A DNA (SEQ ID NO2) is then cleavedwith PstI+XbaI restriction enzymes and inserted into the a similarlycleaved pMAL vector to createpMAL-LC/A-GS18-EN-CPGA16-GS20-H_(N)/A-XbaI-His-tag-stop codon-HindIII.The final construct contains the GS18-EN-CPGA16-GS20 spacer ORF forexpression as a protein of the sequence illustrated in SEQ ID NO10.

Activation Assay

NuPAGE 4-12% Bis-Tris gels (10, 12 and 15 well pre-poured gel) were usedto analyze activation of fusion proteins after treatment with protease.Protein samples were prepared with NuPAGE 4×LDS sample buffer, typicallyto a final volume of 100 μl. Samples were either diluted or made up neat(75 μl of sample, 25 μl of sample buffer) depending on protesinconcentration. The samples were mixed and then heated in the heat blockat 95° C. for 5 min before loading onto the gel. 5-20 μl of sample wasloaded along with 5 μl of the protein marker (Benchmark™ protein markerfrom Invitrogen). The gels were typically run for 50 min at 200 V. Thegel was immersed in dH₂O and microwaved for 2 min on full power. The gelwas rinsed and the microwave step was repeated. The gel was transferredto a staining box and immersed in Simply Blue SafeStain (Invitrogen). Itwas microwaved for 1 minute on full power and left for 0.5-2 h to stain.The gel was then destained by pouring off the Safestain and rinsing thegel with dH₂O. The gels were left in dH₂O to destain overnight and animage was taken on a GeneGnome (Syngene) imager. Total activated proteinwas calculated by comparing the density of the band that corresponded tofull-length fusion protein (after protease treatment) in non-reduced andreduced conditions.

Example 2—Preparation of an LC/A-GS18-EN-CPGA16-GS20-H_(N)/A FusionProtein Family with Variable Spacer Length

Using the same strategy as employed in Example 1, a range of DNA linkerswere prepared that encoded galanin16 and variable spacer content. Usingone of a variety of reverse translation software tools [for exampleEditSeq best E. coli reverse translation (DNASTAR Inc.), orBacktranslation tool v2.0 (Entelechon)], the DNA sequence encoding theSpacer 1-Protease site—ligand-spacer 2 region is determined. Restrictionsites are then incorporated into the DNA sequence and can be arranged asBamHI-SalI-Spacer 1-protease site-CPGA16-NheI-spacer2-SpeI-PstI-XbaI-stop codon-HindIII. It is important to ensure thecorrect reading frame is maintained for the spacer, GA16 and restrictionsequences and that the XbaI sequence is not preceded by the bases, TCwhich would result on DAM methylation. The DNA sequence is screened forrestriction sequence incorporation and any additional sequences areremoved manually from the remaining sequence ensuring common E. colicodon usage is maintained. E. coli codon usage is assessed by referenceto software programs such as Graphical Codon Usage Analyser (Geneart),and the % GC content and codon usage ratio assessed by reference topublished codon usage tables (for example GenBank Release 143, 13 Sep.2004). This optimised DNA sequence is then commercially synthesized (forexample by Entelechon, Geneart or Sigma-Genosys) and is provided in thepCR 4 vector.

The spacer-linkers that were created included:

Spacer 1 - SEQ ID NO of the protease site-GA16 - Spacer 2 linkerGS5-EN-CPGA16-GS20 12, 13, 14, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29 GS10-EN-CPGA16-GS20 33, 34 GS5-EN-CPGA16-HX27 30, 31, 32GS5-EN-CPGA16-GS15 35, 36 GS5-EN-CPGA16-GS10 37, 38 GS18-EN-CPGA16-HX2739, 40 GS18-EN-CPGA16-GS15 41, 42 GS18-EN-CPGA16-GS10 43, 44GS10-EN-CPGA16-HX27 45, 46 GS10-EN-CPGA16-GS15 47, 48GS10-EN-CPGA16-GS10 49, 50

By way of example, in order to create theLC/A-GS5-EN-CPGA16-GS20-H_(N)/A fusion construct (SEQ ID NO12), the pCR4 vector encoding BamHI-SalI-GS5-protease site-GS20-PstI-XbaI-stopcodon-HindIII the linker is cleaved with BamHI+SalI restriction enzymes.This cleaved vector then serves as the recipient vector for insertionand ligation of the LC/A DNA (SEQ ID NO1) also cleaved with BamHI+SalI.The resulting plasmid DNA is then cleaved with BamHI+HindIII restrictionenzymes and the LC/A-linker fragment inserted into a similarly cleavedvector containing a unique multiple cloning site for BamHI, SalI, PstI,and HindIII such as the pMAL vector (NEB) or the pET vector (Novagen).The H_(N)/A DNA (SEQ ID NO2) is then cleaved with PstI+HindIIIrestriction enzymes and inserted into the similarly cleavedpMAL-LC/A-linker construct. The final construct contains theLC/A-GS5-EN-CPGA16-GS20-H_(N)/A ORF for expression as a protein of thesequence illustrated in SEQ ID NOl13.

Example 3—Purification Method for Galanin Fusion Protein

Defrost falcon tube containing 25 ml 50 mM HEPES pH 7.2, 200 mM NaCl andapproximately 10 g of E. coli BL21 cell paste. Make the thawed cellpaste up to 80 ml with 50 mM HEPES pH 7.2, 200 mM NaCl and sonicate onice 30 seconds on, 30 seconds off for 10 cycles at a power of 22 micronsensuring the sample remains cool. Spin the lysed cells at 18 000 rpm, 4°C. for 30 minutes. Load the supernatant onto a 0.1 M NiSO₄ chargedChelating column (20-30 ml column is sufficient) equilibrated with 50 mMHEPES pH 7.2, 200 mM NaCl. Using a step gradient of 10 and 40 mMimidazole, wash away the non-specific bound protein and elute the fusionprotein with 100 mM imidazole. Dialyse the eluted fusion protein against5 L of 50 mM HEPES pH 7.2, 200 mM NaCl at 4° C. overnight and measurethe OD of the dialysed fusion protein. Add 1 μg of enterokinase (1mg/ml) per 100 μg of purified fusion protein and 10 μl of factor Xa permg of purified fusion protein if the fusion protesin contains a maltosebinding protein. Incubate at 25° C. static overnight. Load onto a 0.1 MNiSO₄ charged Chelating column (20-30 ml column is sufficient)equilibrated with 50 mM HEPES pH 7.2, 200 mM NaCl. Wash column tobaseline with 50 mM HEPES pH 7.2, 200 mM NaCl. Using a step gradient of10 and 40 mM imidazole, wash away the non-specific bound protein andelute the fusion protein with 100 mM imidazole. Dialyses the elutedfusion protein against 5 L of 50 mM HEPES pH 7.2, 200 mM NaCl at 4° C.overnight and concentrate the fusion to about 2 mg/ml, aliquot sampleand freeze at −20° C.

Example 4—Preparation of a LC/C-GA16-H_(N)/IC Fusion Protein with aSerotype A Activation Sequence

Following the methods used in Examples 1 and 2, the LC/C (SEQ ID NO5)and H_(N)/C (SEQ ID NO6) are created and inserted into the A serotypelinker arranged as BamHI-SalI-Spacer 1-protease site-GA16-NheI-spacer2-SpeI-PstI-XbaI-stop codon-HindIII. The final construct contains theLC-spacer 1-GA16-spacer 2-H_(N) ORF for expression as a protein of thesequence illustrated in SEQ ID NO25.

Example 5—Preparation of an IgA Protease-GA16-H_(N)/A Fusion Protein

The IgA protease amino acid sequence was obtained from freely availabledatabase sources such as GenBank (accession number P09790). Informationregarding the structure of the N. gonorrhoeae IgA protease gene isavailable in the literature (Pohlner et al., Gene structure andextracellular secretion of Neisseria gonorrhoeae IgA protease, Nature,1987, 325(6103), 458-62). Using Backtranslation tool v2.0 (Entelechon),the DNA sequence encoding the IgA protease modified for E. coliexpression was determined. A BamHI recognition sequence was incorporatedat the 5′ end and a codon encoding a cysteine amino acid and SalIrecognition sequence were incorporated at the 3′ end of the IgA DNA. TheDNA sequence was screened using MapDraw, (DNASTAR Inc.) for restrictionenzyme cleavage sequences incorporated during the back translation. Anycleavage sequences that are found to be common to those required forcloning were removed manually from the proposed coding sequence ensuringcommon E. coli codon usage is maintained. E. coli codon usage wasassessed Graphical Codon Usage Analyser (Geneart), and the % GC contentand codon usage ratio assessed by reference to published codon usagetables. This optimised DNA sequence (SEQ ID NO51) containing the IgAopen reading frame (ORF) is then commercially synthesized.

The IgA (SEQ ID NO51) is inserted into the LC-GS5-CPGA16-GS20-H_(N) ORFusing BamHI and SalI restriction enzymes to replace the LC with the IgAprotease DNA. The final construct contains the IgA-GS5-CPGA16-GS20-H_(N)ORF for expression as a protein of the sequence illustrated in SEQ IDNO53.

Example 6—Preparation of a Galanin Targeted Endopeptidase Fusion ProteinContaining a LC Domain Derived from Tetanus

The DNA sequence is designed by back translation of the tetanus toxin LCamino acid sequence (obtained from freely available database sourcessuch as GenBank (accession number X04436) using one of a variety ofreverse translation software tools [for example EditSeq best E. colireverse translation (DNASTAR Inc.), or Backtranslation tool v2.0(Entelechon)]. BamHI/SalI recognition sequences are incorporated at the5′ and 3′ ends respectively of the sequence maintaining the correctreading frame (SEQ ID NO57). The DNA sequence is screened (usingsoftware such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavagesequences incorporated during the back translation. Any cleavagesequences that are found to be common to those required by the cloningsystem are removed manually from the proposed coding sequence ensuringcommon E. coli codon usage is maintained. E. coli codon usage isassessed by reference to software programs such as Graphical Codon UsageAnalyser (Geneart), and the % GC content and codon usage ratio assessedby reference to published codon usage tables (for example GenBankRelease 143, 13 Sep. 2004). This optimised DNA sequence containing thetetanus toxin LC open reading frame (ORF) is then commerciallysynthesized (for example by Entelechon, Geneart or Sigma-Genosys) and isprovided in the pCR 4 vector (invitrogen). The pCR 4 vector encoding theTeNT LC is cleaved with BamHI and SalI. The BamHI-SalI fragment is theninserted into the LC/A-GA16-H_(N)/A vector that has also been cleaved byBamHI and SalI. The final construct contains the TeNTLC-GS5-GA16-GS20-H_(N) ORF sequences for expression as a protein of thesequence illustrated in SEQ ID NO58.

Example 7—Construction of CHO-K1 GALR1 & GALR2 Receptor Activation Assayand SNAP-25 Cleavage Assay

Cell-Line Creation

CHO-K1 cells stably expressing either the human galanin 1 receptor(CHO-K1-Gal-1R; product number ES-510-C) or human galanin 2 receptor(CHO-K1-Gal-2R; product number ES-511-C) were purchased fromPerkin-Elmer (Bucks, UK). Where required, cells were transfected withSNAP-25 DNA using Lipofectamine™ 2000 and incubated for 4 hours beforemedia replacement. After 24 hours, cells were transferred to a T175flask. 100 ug/ml Zeocin was added after a further 24 hours to beginselection of SNAP-25 expressing cells, and 5 ug/ml Blasticidin added tomaintain selective pressure for the receptor. Cells were maintained inmedia containing selection agents for two weeks, passaging cells everytwo to three days to maintain 30-70% confluence. Cells were then dilutedin selective media to achieve 0.5 cell per well in a 96 well microplate.After a few days, the plates were examined under a microscope, and thosecontaining single colonies were marked. Media in these wells was changedweekly. As cells became confluent in the wells, they were transferred toT25 flasks. When they had expanded sufficiently each clone was seeded to24 wells of a 96 well plate, plus a frozen stock vial created. Galaninfusion proteins of the invention and LC/A-H_(N)A were applied to thecells for 24 hours, and then western blots performed to detect SNAP-25cleavage. Clones from which SNAP-25 bands were strong and cleavagelevels were high with fusion were maintained for further investigation.Full dose curves were run on these, and the clone with the highestdifferential between galanin fusion protein and LC/A-H_(N)A cleavagelevels was selected.

GALR1 Receptor Activation Assay

The GALR1 receptor activation assay measures the potency and intrinsicefficacy of ligands at the GALR1 receptor in transfected CHO-K1 cells byquantifying the reduction of forskolin-stimulated intracellular cAMPusing a FRET-based cAMP (Perkin Elmer LANCE cAMP kit). Afterstimulation, a fluorescently labelled cAMP tracer(Europium-streptavadin/biotin-cAMP) and fluorescently (Alexa) labelledanti-cAMP antibody are added to the cells in a lysis buffer. cAMP fromthe cells competes with the cAMP tracer for antibody binding sites. Whenread, a light pulse at 320 nm excites the fluorescent portion (Europium)of the cAMP tracer. The energy emitted from the europium is transferredto the Alexa fluor-labelled antibodies bound to the tracer, generating aTR-FRET signal at 665 nm (Time-resolved fluorescence resonance energytransfer is based on the proximity of the donor label, europium, and theacceptor label, Alexa fluor, which have been brought together by aspecific binding reaction). Residual energy from the europium produceslight at 615 nm. In agonist treated cells there will be less cAMP tocompete with the tracer so a dose dependant increase in signal at 665 nmwill be observed compared with samples treated with forskolin alone. Thesignal at 665 nm signal is converted to cAMP concentration byinterpolation to a cAMP standard curve which is included in eachexperiment.

Using Gilson pipettes and Sigmacoted or lo-bind tips, test materials andstandards were diluted to the appropriate concentrations in the wells ofthe first two columns of an eppendorf 500 μl deep-well lo-bind plate, inassay buffer containing 10 μM forskolin. The chosen concentrations incolumns one and two were half a log unit apart. From these, serial 1:10dilutions were made across the plate (using an electronic eight channelpipette with sigmacote or lo-bind tips) until eleven concentrations athalf log intervals had been created. In the twelfth column, assay bufferonly was added as a ‘basal’. Using a 12 channel digital pipette, 10 μlof sample from the lo-bind plate was transferred to the optiplate 96well microplate.

To wells containing the standard curve, 10 ul of assay buffer was addedusing a multichannel digital pipette. To wells containing the testmaterials, 10 ul of cells in assay buffer at the appropriateconcentration were added. Plates were sealed and incubated for 120 minat room temperature, for the first hour on an IKA MTS 2/4 orbital shakerset to maximum speed.

LANCE Eu-W8044 labelled streptavidin (Eu-SA) and Biotin-cAMP (b-cAMP)were diluted in cAMP Detection Buffer (both from Perkin Elmer LANCE cAMPkit) to create sub-stocks, at dilution ratios of 1:17 and 1:5,respectively. The final detection mix was prepared by diluting from thetwo sub stocks into detection buffer at a ratio of 1:125. The mixturewas incubated for 15-30 min at room temperature before addition of 1:200Alexa Fluor® 647-anti cAMP Antibody (Alexa-Fluor Ab). After brieflyvortex mixing, 20 μl was immediately added to each well using a digitalmultichannel pipette. Microplate sealers were applied and platesincubated for 24 h at room temperature (for the first hour on an IKA MTS2/4 orbital shaker set to maximum speed). Plate sealers were removedprior to reading on the Envision.

GALR2 Receptor Activation Assay

The GALR2 receptor activation assay measures the potency and intrinsicefficacy of ligands at GALR2 receptor in transfected CHO-K1 cells bymeasuring the calcium mobilisation that occurs when the receptor isactivated. The transfected cells are pre-loaded with a calcium sensitivedye (FLIPR) before treatment. When read using Flexstation 3 microplatereader (Molecular devices) a light pulse at 485 nm excites thefluorescent dye and causes an emission at 525 nm. This providesreal-time fluorescence data from changes in intracellular calcium. Inagonist treated cells there will be activation of the receptor, leadingto an increase in calcium mobilisation. This will be measured as anincrease in the relative fluorescence units (RFU) at 525 nM.

Culture of Cells for Receptor Activation Assay:

Cells were seeded and cultured in T175 flasks containing Ham F12 withGlutamax, 10% Foetal bovine serum, 5 μg ml-1 Blasticidin and 100 μg ml-1Zeocin. The flasks were incubated at 37° C. in a humidified environmentcontaining 5% CO₂ until 60-80% confluent. On the day of harvest themedia was removed and the cells washed twice with 25 ml PBS. The cellswere removed from the flask by addition of 10 ml of Tryple Express, andincubation at 37° C. for 10 min followed by gentle tapping of the flask.The dislodged cells were transferred to a 50 ml centrifuge tube and theflask washed twice with 10 ml media which was added to the cellsuspension. The tube was centrifuged at 1300×g for 3 min and thesupernatant removed. Cells were gently re-suspended in 10 ml media (iffreezing cells) or assay buffer (if using ‘fresh’ cells in assay), and asample was removed for counting using a nucleocounter (ChemoMetec).Cells for use ‘fresh’ in an assay were diluted further in assay bufferto the appropriate concentration. Cells harvested for freezing werere-centrifuged (1300×g; 3 min), the supernatant removed and cellsre-suspended in Synth-a-freeze at 4° C. to 3×106 cells/ml. Cryovialscontaining 1 ml suspension each were placed in a chilled Nalgene MrFrosty freezing container (−1° C./minute cooling rate), and leftovernight in a −80° C. freezer. The following day vials were transferredto the vapour phase of a liquid nitrogen storage tank.

FIG. 4 demonstrates that galanin fusion proteins of the presentinvention having different galanin ligands (i.e. galanin-16 andgalanin-30) and different serotype backbones (i.e. LC/A-H_(N)/A,LC/B-H_(N)/B, LC/C-H_(N)/C and LC/D-H_(N)/D) activate GALR1 receptors.

CHO-K1 GALR1 SNAP-25 Cleavage Assays

Cultures of cells were exposed to varying concentrations of galaninfusion protein for 24 hours. Cellular proteins were separated bySDS-PAGE and western blotted with anti-SNAP-25 antibody to facilitateassessment of SNAP-25 cleavage. SNAP-25 cleavage calculated bydensitometric analysis (Syngene).

Plating Cells

Prepare cells at 2×10e5 cells/ml and seed 125 μl per well of 96 wellplate. Use the following media: 500 ml Gibco Ham F12 with Glutamax(product code 31765068), 50 ml FBS, 5 ug/ml Blasticidin (250 μl aliquotfrom box in freezer, G13) (Calbiochem #203351, 10 ml at 10 mg/ml), 100ug/ml Zeocin (500 μl from box in freezer, G35). (Invitrogen from Fisher,1 g in 8×1.25 ml tubes at 100 mg/ml product code VXR25001). Allow cellsto grow for 24 hrs (37° C., 5% CO₂, humidified atmosphere).

Cell Treatment

Prepare dilutions of test protein for a dose range of each test proteins(make up double (2×) the desired final concentrations because 125 μlwill be applied directly onto 125 μl of media already in each well).Filter sterilize CHO GALR1 feeding medium (20 ml syringe, 0.2 μm syringefilter) to make the dilutions. Add the filtered medium into 5 labelledbijoux's (7 ml tubes), 0.9 ml each using a Gilson pipette ormulti-stepper. Dilute the stock test protein to 2000 nM (working stocksolution 1) and 600 nM (working stock solution 2). Using a Gilsonpipette prepare 10-fold serial dilutions of each working stock, byadding 100 μl to the next concentration in the series. Pipette up anddown to mix thoroughly. Repeat to obtain 4 serial dilutions for solution1, and 3 serial dilutions for solution 2. A 0 nM control (filteredfeeding medium only) should also be prepared as a negative control foreach plate. Repeat the above for each test protein. In each experiment a‘standard’ batch of material must be included as control/referencematerial, this is unliganded LC/A-H_(N)/A.

Apply Diluted Sample to CHO GALR1 Plates

Apply 125 μl of test sample (double concentration) per well. Each testsample should be applied to triplicate wells and each dose range shouldinclude a 0 nM control. Incubate for 24 hrs (37° C., 5% CO₂, humidifiedatmosphere).

Cell Lysis

Prepare fresh lysis buffer (20 mls per plate) with 25% (4×) NuPAGE LDSsample buffer, 65% dH₂O and 10% 1 M DTT. Remove medium from the CHOGALR1 plate by inverting over a waste receptacle. Drain the remainingmedia from each well using a fine-tipped pipette. Lyse the cells byadding 125 μl of lysis buffer per well using a multi-stepper pipette.After a minimum of 20 mins, remove the buffer from each well to a 1.5 mlmicrocentrifuge tube. Tubes must be numbered to allowing tracking of theCHO GALR1 treatments throughout the blotting procedure. A1-A3 down toH1-H3 numbered 1-24, A4-A6 down to H4-H6 numbered 25-48, A7-A9 down toH7-H93 numbered 49-72, A10-A12 down to H10-H12 numbered 73-96. Vortexeach sample and heat at 90° C. for 5-10 mins in a prewarmed heat block.Store at −20° C. or use on the same day on an SDS gel.

Gel Electrophoresis

If the sample has been stored o/n or longer, put in a heat blockprewarmed to 90° C. for 5-10 mins. Set up SDS page gels, use 1 gel per12 samples, prepare running buffer (1×, Invitrogen NuPAGE MOPS SDSRunning Buffer (20×) (NP0001))≈800 ml/gel tank. Add 500 μl of NuPAGEantioxidant to the upper buffer chamber. Load 15 ul samples onto gellanes from left to right as and load 2.5 ul of Invitrogen Magic MarkerXP and 5 ul Invitrogen See Blue Plus 2 pre-stained standard and 15 ul ofnon-treated control. It is important to maximize the resolution ofseparation during SDS_PAGE. This can be achieved by running 12% bis-trisgels at 200 V for 1 hour and 25 minutes (until the pink (17 kDa) markerreaches the bottom of the tank).

Western Blotting

Complete a Semi-dry transfer: using an Invitrogen iBlot (use iBlotProgramme 3 for 6 minutes). Put the nitrocellulose membranes inindividual small trays. Incubate the membranes with blocking buffersolution (5 g Marvel milk powder per 100 ml 0.1% PBS/Tween) at roomtemperature, on a rocker, for 1 hour. Apply primary antibody(Anti-SNAP-25 1:1000 dilution) and incubate the membranes with primaryantibody (diluted in blocking buffer) for 1 hour on a rocker at roomtemperature. Wash the membranes by rinsing 3 times with PBS/Tween(0.1%). Then apply the secondary (Anti-Rabbit-HRP conjugate diluted1:1000) and incubate the membranes with secondary antibody (diluted inblocking buffer) at room temperature, on a rocker, for 1 hour. Wash themembranes by rinsing 3 times with PBS/Tween (0.1%), leave membrane aminimum of 20 mins for the last wash. Detect the bound antibody usingSyngene: Drain blots of PBS/Tween, mix WestDura reagents 1:1 and add toblots for 5 minutes. Ensure enough solution is added to the membranes tocompletely cover them. Place membrane in Syngene tray, set up Syngenesoftware for 5 min expose time.

FIGS. 3 and 5 demonstrate that galanin fusion proteins of the inventioneffectively cleave SNAP-25.

Example 8—Assessment of In Vivo Efficacy of a Galanin Fusion

The nociceptive flexion reflex (also known as paw guarding assay) is arapid withdrawal movement that constitutes a protective mechanismagainst possible limb damage. It can be quantified by assessment ofelectromyography (EMG) response in anesthetized rat as a result of lowdose capsaicin, electrical stimulation or the capsaicin-sensitizedelectrical response. Intraplantar pretreatment (24 hour) of fusionproteins of the present invention into 300-380 g male Sprague-Dawleyrats. Induction of paw guarding was achieved by 0.006% capsaicin, 10 μlin PBS (7.5% DMSO), injected in 10 seconds. This produced a robustreflex response from biceps feroris muscle. A reduction/inhibition ofthe nociceptive flexion reflex indicates that the test substancedemonstrates an antinociceptive effect. The data demonstrated theantinociceptive effect of the galanin fusion proteins of the presentinvention as a percentage (FIG. 6)

The ability of different galanin fusion proteins of the invention toinhibit capsaicin-induced thermal hyperalgesia was evaluated (FIGS. 7and 8). Intraplantar pretreatment of fusion proteins into Sprague-Dawleyrats and 24 hours later 0.3% capsaicin was injected and rats were put on25° C. glass plate (rats contained in acrylic boxes, on 25° C. glassplate). Light beam (adjustable light Intensity) focused on the hind paw.Sensors detected movement of paw, stopping timer. Paw Withdrawal Latencyis time to remove paw from heat source (Cut-off of 20.48 seconds). Areduction/inhibition of the paw withdrawal latency indicates that thetest substance demonstrates an antinociceptive effect. The datademonstrated the enhanced antinociceptive effect of the galanin fusionproteins of the present invention compared to fusion proteins with aC-terminally presented ligand.

Example 9—Confirmation of TM Agonist Activity by Measuring Release ofSubstance P from Neuronal Cell Cultures

Materials

Substance P EIA is obtained from R&D Systems, UK.

Methods

Primary neuronal cultures of eDRG are established as describedpreviously (Duggan et al., 2002). Substance P release from the culturesis assessed by EIA, essentially as described previously (Duggan et al.,2002). The TM of interest is added to the neuronal cultures (establishedfor at least 2 weeks prior to treatment); control cultures are performedin parallel by addition of vehicle in place of TM. Stimulated (100 mMKCl) and basal release, together with total cell lysate content, ofsubstance P are obtained for both control and TM treated cultures.Substance P immunoreactivity is measured using Substance P EnzymeImmunoassay Kits (Cayman Chemical Company, USA or R&D Systems, UK)according to manufacturers' instructions.

The amount of Substance P released by the neuronal cells in the presenceof the TM of interest is compared to the release obtained in thepresence and absence of 100 mM KCl. Stimulation of Substance P releaseby the TM of interest above the basal release, establishes that the TMof interest is an “agonist ligand” as defined in this specification. Ifdesired the stimulation of Substance P release by the TM of interest canbe compared to a standard Substance P release-curve produced using thenatural ORL-1 receptor ligand, nociceptin (Tocris).

Example 10

A method of treating, preventing or ameliorating pain in a subject,comprising administration to said patient a therapeutic effective amountof fusion protein, wherein said pain is selected from the groupconsisting of: chronic pain arising from malignant disease, chronic painnot caused by malignant disease (peripheral neuropathies).

Patient A

A 73 year old woman suffering from severe pain caused by posthepaticneuralgia is treated by a peripheral injection with fusion protein toreduce neurotransmitter release at the synapse of nerve terminals toreduce the pain. The patient experiences good analgesic effect within 2hours of said injection.

Patient B

A 32 year old male suffering from phantom limb pain after having hisleft arm amputated following a car accident is treated by peripheralinjection with fusion protein to reduce the pain. The patientexperiences good analgesic effect within 1 hour of said injection.

Patient C

A 55 year male suffering from diabetic neuropathy is treated by aperipheral injection with fusion protein to reduce neurotransmitterrelease at the synapse of nerve terminals to reduce the pain. Thepatient experiences good analgesic effect within 4 hours of saidinjection.

Patient D

A 63 year old woman suffering from cancer pain is treated by aperipheral injection with fusion protein to reduce neurotransmitterrelease at the synapse of nerve terminals to reduce the pain. Thepatient experiences good analgesic effect within 4 hours of saidinjection.

All documents, books, manuals, papers, patents, published patentapplications, guides, abstracts and other reference materials citedherein are incorporated by reference in their entirety. While theforegoing specification teaches the principles of the present invention,with examples provided for the purpose of illustration, it will beappreciated by one skilled in the art from reading this disclosure thatvarious changes in form and detail can be made without departing fromthe true scope of the invention.

1. A single chain, polypeptide fusion protein, comprising: a. anon-cytotoxic protease, which protease cleaves a protein of the exocyticfusion apparatus of a nociceptive sensory afferent; b. a galaninTargeting Moiety that binds to a Binding Site on the nociceptive sensoryafferent, which Binding Site endocytoses to be incorporated into anendosome within the nociceptive sensory afferent; c. a protease cleavagesite at which site the fusion protein is cleavable by a protease,wherein the protease cleavage site is located between the non-cytotoxicprotease and the galanin Targeting Moiety; d. a translocation domainthat translocates the protease from within an endosome, across theendosomal membrane and into the cytosol of the nociceptive sensoryafferent, wherein the Targeting Moiety is located between the proteasecleavage site and the translocation domain; e. a first spacer locatedbetween the non-cytotoxic protease and the protease cleavage site,wherein said first spacer comprises an amino acid sequence of from 4 to25 amino acid residues; f. a second spacer located between the galaninTargeting Moiety and the translocation domain, wherein said secondspacer comprises an amino acid sequence of from 4 to 35 amino acidresidues.
 2. The fusion protein according to claim 1, wherein the firstspacer comprises an amino acid sequence of from 6 to 16 amino acidresidues.
 3. The fusion protein according to claim 1 or claim 2, whereinsaid amino acid residues of said first spacer are selected from thegroup consisting of glycine, threonine, arginine, serine, alanine,asparagine, glutamine, aspartic acid, proline, glutamic acid and/orlysine.
 4. The fusion protein according to any of claims 1-3, whereinthe amino acid residues of the first spacer are selected from the groupconsisting of glycine, serine and alanine.
 5. The fusion proteinaccording to any of claims 1-4, wherein the first spacer is selectedfrom a GS5, GS10, GS15, GS18 or GS20 spacer.
 6. The fusion proteinaccording to any of claims 1-5, wherein the galanin Targeting Moietybinds specifically to the GALR1, GALR2 and/or the GALR3 receptor.
 7. Thefusion protein according to any of claim 1-6, wherein the galaninTargeting Moiety comprises or consists of an amino acid sequence havingat least 70% sequence identity to SEQ ID NO: 7 or SEQ ID NO:
 8. 8. Thefusion protein according to any of claims 1-7, wherein the galaninTargeting Moiety comprises an amino acid sequence according to SEQ IDNO. 7 or a fragment comprising or consisting of at least 14 or 16contiguous amino acid residues thereof, or a variant amino acid sequenceof said SEQ ID NO: 7 or said fragment having a maximum of 5 or 6conservative amino acid substitutions.
 9. The fusion protein accordingto any of claims 1-8, wherein the non-cytotoxic protease is aclostridial neurotoxin L-chain or an IgA protease.
 10. The fusionprotein according to any of claims 1-9, wherein the translocation domainis the H_(N) domain of a clostridial neurotoxin.
 11. The fusion proteinaccording to any of claims 1-10, wherein said fusion protein comprisesan amino acid sequence having at least 90% sequence identity to theamino acid sequence selected from the group consisting of SEQ ID NOs:10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 53, 56and/or
 59. 12. A polynucleotide molecule encoding the polypeptide fusionprotein according to any of claims 1-11.
 13. An expression vector, whichcomprises a promoter, the polynucleotide molecule according to claim 12,wherein said polynucleotide molecule is located downstream of thepromoter, and a terminator located downstream of the polynucleotidemolecule.
 14. A method for preparing a single-chain polypeptide fusionprotein, comprising: a. transfecting a host cell with the expressionvector of claim 13, and b. culturing said host cell under conditionspromoting expressing of the polypeptide fusion protein by the expressionvector.
 15. A method of preparing a non-cytotoxic agent, comprising: a.contacting a single-chain polypeptide fusion protein according to any ofclaims 1-11 with a protease capable of cleaving the protease cleavagesite; b. cleaving the protease cleavage site; and thereby forming adi-chain fusion protein.
 16. A non-cytotoxic polypeptide, obtained bythe method of claim 15, wherein the polypeptide is a di-chainpolypeptide, and wherein: a. the first chain comprises the non-cytotoxicprotease, which protease is capable of cleaving a protein of theexocytic fusion apparatus of a nociceptive sensory afferent; b. thesecond chain comprises the galanin TM and the translocation domain thatis capable of translocating the protease from within an endosome, acrossthe endosomal membrane and into the cytosol of the nociceptive sensoryafferent; and the first and second chains are disulphide linkedtogether.
 17. A method of treating, preventing or ameliorating pain in asubject, comprising administering to said patient a therapeuticallyeffective amount of the fusion protein according to any of claims 1-11.18. A method according to claim 17, wherein the pain is chronic painselected from neuropathic pain, inflammatory pain, headache pain,somatic pain, visceral pain, and referred pain.
 19. A method oftreating, preventing or ameliorating pain in a subject, comprisingadministering to said patient a therapeutically effective amount of apolypeptide according to claim
 16. 20. A method according to claim 19,wherein the pain is chronic pain selected from neuropathic pain,inflammatory pain, headache pain, somatic pain, visceral pain, andreferred pain.